Enzyme-catalyzed enantioselective aziridination of olefins

ABSTRACT

The present invention provides methods for catalyzing the conversion of an olefin to a compound containing one or more aziridine functional groups using heme enzymes. In certain aspects, the present invention provides a reaction mixture for producing an aziridination product, the reaction mixture comprising of an olefinic substrate, a nitrene precursor, and a heme enzyme. In other certain aspects, the present invention provides a method for producing an aziridination product comprising providing an olefinic substrate, a nitrene precursor, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce an aziridine product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin aziridination reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 62/108,300, filed Jan. 27, 2015, and U.S. ProvisionalPatent Application No. 62/120,126, filed Feb. 24, 2015, the contents ofwhich are hereby incorporated by reference in their entirety for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.N00014-11-1-0205 awarded by the Office of Naval Research. The governmenthas certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file086544-019120US-0966192_SequenceListing.txt, created on Apr. 11, 2016,418,993 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Aziridines are 3 membered cyclic compounds comprising 2 carbons and anitrogen that are often used as building blocks in various syntheticstrategies. Traditional synthesis of aziridines can be achieved throughvarious known methods; however, many of these method use causticchemicals, harsh reaction conditions, and/or are unable to producestereo-selective chiral aziridines.

Enzymes offer appealing alternatives to traditional chemical catalystsdue to their ability to function in aqueous media at ambient temperatureand pressure, as well as their ability to orient substrate binding fordefined regio- and stereo-chemical outcomes. Indeed, the use of enzymesin synthetic chemistry to achieve otherwise difficult or low yieldingchemical conversions is continuing to increase.

Although chemically attractive, enzymes are also known for their highsubstrate specificity and their catalytic fidelity. While thisselectivity can be advantageous in some cases, it is also a significantsynthetic limitation because each enzyme typically catalyzes only aspecific chemical reaction. Despite these limitations, previous studieshave shown that the native activity of enzymes can be modified oraltered to catalyze non-natural or non-naturally occurring chemicalconversions. Development of an enzyme capable of catalyzing anaziridination reaction could avoid using caustic chemicals, harshreaction conditions, and could reliably produce stereo-selective chiralaziridines.

As such, there is a need in the art for novel reagents and catalyticschemes that are capable of creating an aziridine functional group withhigh yield, regioselectivity, and stereoselectivity, but without thetoxicity and harsh reaction conditions associated with currentapproaches. The present invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides a reaction mixture forproducing an aziridination product. The reaction mixture includes anolefinic substrate, a nitrene precursor, and a heme enzyme.

In some embodiments, the olefinic substrate is represented by astructure of Formula I:

wherein:

-   -   R^(1a), R^(1b), and R² are independently selected from the group        consisting of H, C₁₋₈alkyl, C₁₋₈heteroalkyl, aryl, heteroaryl,        C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl, —Y¹-heteroaryl,        —Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl;    -   Y¹ is C₁₋₈alkylene;    -   each R^(1a), R^(1b), and R² is optionally substituted with from        1 to 5 substituents independently selected from the group        consisting of C₁₋₃alkyl, alkoxy hydroxyl, amino, thiol, carboxy,        amido, oxo, thioxo, cyano, and halogen;    -   wherein each aryl contains between 6-14 carbon atoms, each        heteroaryl group has from 5 to 8 ring atoms and from 1-3        heteroatoms selected from N, O and S, and each heterocyclyl        group has from 1-3 heteroatoms selected from N, O and S.

In some instances, R^(1a), R^(1b), and R² are independently selectedfrom the group consisting of H, C₁₋₁₈alkyl, aryl, heteroaryl,C₁₋₁₂cycloalkyl, and C₃₋₁₀heterocyclyl, and each R^(1a), R^(1b), and R²is optionally substituted with from 1 to 5 substituents independentlyselected from the group consisting of C₁₋₃alkyl, alkoxy, and halogen.

In some embodiments, the nitrene precursor has a structure according toFormula IIa or IIb:

wherein:

-   -   R³ is selected from the group consisting of C₁₋₁₈ alkyl,        C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,        C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b),        —PO₃R^(b)R^(c), and —CONR^(b)R^(c);    -   X¹ is independently selected from the group consisting of H and        sodium, and    -   X² is independently selected from the group consisting of        halogen, —SO₂R^(a), —CO₂R^(b), —PO₃R^(b)R^(c), optionally X¹ and        X² can be taken together to form iodinane;    -   R^(a) is independently selected from the group consisting of        C₁₋₈alkyl, hydroxy, C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl,        heteroaryl, and C₃₋₈heterocyclyl;    -   R^(b) and R^(c) are independently selected from the group        consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl, and        C₃₋₈heterocyclyl;    -   wherein within each R³, R^(a), R^(b), and R^(c) can be        optionally substituted with from 1-5 R^(d) substituents;    -   each R^(d) is independently selected from the group consisting        of C₁₋₃alkyl, halogen, and hydroxy; and    -   wherein each aryl contains between 6-14 carbon atoms, each        heteroaryl group has from 5 to 10 ring atoms and from 1-3        heteroatoms selected from N, O and S, and each heterocyclyl        group has from 1-3 heteroatoms selected from N, O and S.

In some embodiments, the nitrene precursor has a structure selected fromthe group consisting of:

In some instances, the nitrene precursor is

In some embodiments, the aziridination product is produced in vitro.

In some embodiments, the reaction mixture further comprises a reducingagent. In some instances, the reducing agent is NADPH.

In some embodiments, the heme the heme enzyme is localized within awhole cell and the aziridination product is produced in vivo. In someinstances, the whole cell is a bacterial cell or a yeast cell.

In some embodiments, the aziridination product is produced underanaerobic conditions.

In some embodiments, the heme enzyme is a variant thereof comprising amutation at the axial position of the heme coordination site. In someinstances, the heme enzyme comprises a serine mutation at the axialposition of the heme coordination site.

In some embodiments, the heme enzyme is a cytochrome P450 enzyme or avariant thereof. In some instances, the cytochrome P450 enzyme is a P450BM3 enzyme or a variant thereof.

In some embodiments, the P450 BM3 enzyme comprises an axial ligandmutation C400S and one or more mutations selected from the groupconsisting of V78, F87, P142, T175, A184, S226, H236, E252, I263, T268,A290, A328, L353, I366, L437, T438, and E442 relative to the amino acidsequence set forth in SEQ ID NO:1 (SEQ ID NO: 50). In some instances,the P450 BM3 enzyme comprises an axial ligand mutation C400S andmutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,T268A, A290V, A328V, L353V, I366V, L437V, T438S, and E442K relative tothe amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 51).

In some embodiments, the P450 BM3 enzyme comprises an axial ligandmutation C400S and one or more mutations selected from the groupconsisting of L75, V78, F87, P142, T175, L181, A184, S226, H236, E252,I263, T268, A290, L353, I366, and E442 relative to the amino acidsequence set forth in SEQ ID NO:1 (SEQ ID NO: 52). In some instances,the P450 BM3 enzyme comprises an axial ligand mutation C400S andmutations L75A, V78A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q,E252G, I263F, T268A, A290V, L353V, I366V, and E442K relative to theamino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 53).

In some embodiments, the aziridination product is an aziridine compoundaccording to Formula III:

wherein

-   -   R^(1a), R^(1b), and R² are independently selected from the group        consisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl, aryl, heteroaryl,        C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl, —Y¹-heteroaryl,        —Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl;    -   Y¹ is C₁₋₈alkylene;    -   each R^(1a), R^(1b), and R² is optionally substituted with from        1 to 5 substituents independently selected from the group        consisting of C₁₋₃alkyl, alkoxy hydroxyl, amino, thiol, carboxy,        amido, oxo, thioxo, cyano, and halogen;    -   R³ is selected from the group consisting of C₁₋₁₈ alkyl,        C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,        C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b),        —PO₃R^(b)R^(c), and —CONR^(b)R^(c);    -   R^(a) is independently selected from the group consisting of        C₁₋₈alkyl, hydroxy, C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl,        heteroaryl, and C₃₋₈heterocyclyl;    -   R^(b) and R^(c) are independently selected from the group        consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl, and        C₃₋₈heterocyclyl;    -   wherein within each R³, R^(a), R^(b), and R^(c) can be        optionally substituted with from 1-5 R^(d) substituents;    -   each R^(d) is independently selected from the group consisting        of C₁₋₃alkyl, halogen, and hydroxy; and    -   wherein each aryl contains between 6-14 carbon atoms, each        heteroaryl group has from 5 to 10 ring atoms and from 1-3        heteroatoms selected from N, O and S, and each heterocyclyl        group has from 1-3 heteroatoms selected from N, O and S.

In some instances, R^(1a) and R^(1b) are independently selected from thegroup consisting of H, C₁₋₈alkyl, aryl, heteroaryl, C₁₋₁₂cycloalkyl, andC₃₋₁₀heterocyclyl;

-   -   R² is selected from the group consisting of H and C₁₋₈ alkyl;    -   each R^(1a), R^(1b), and R² is optionally substituted with from        1 to 3 substituents independently selected from the group        consisting of C₁₋₃alkyl, alkoxy, and halogen; and    -   R³ is selected from the group consisting of —SO₂R^(a), —COR^(a),        —CO₂R^(b), —PO₃R^(b)R^(c), and —CONR^(b)R^(c),    -   R^(a) is independently selected from the group consisting of        C₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl;    -   R^(b) and R^(c) are independently selected from the group        consisting of C₃₋₁₂cycloalkyl, aryl, heteroaryl, and        C₃₋₈heterocyclyl;    -   wherein within each R³, R^(a), R^(b), and R^(c) can be        optionally substituted with from 1-2 R^(d) substituents; and    -   each R^(d) is independently selected from the group consisting        of C₁₋₃alkyl, halogen, and hydroxy.

In some embodiments, the aziridination product is an amido-alcoholcompound according for Formula IIIa:

wherein R^(1a), R^(1b), R³, and R³ can be as defined above in FormulaIII.

In some embodiments, the reaction produces a plurality of aziridinationproducts. In some instances, the plurality of aziridination products hasa % ee_(S) of from about −99% to about 99%. In some instances, theplurality of aziridination products has a % ee_(S) of from about −81% toabout 81%. In some instances, the plurality of aziridination productshas a Z:E ratio of from about 1:99 to about 99:1.

In some aspects, the present invention provides a cytochrome P450 BM3enzyme variant or fragment thereof that can a aziridinate an olefinicsubstrate comprising an axial ligand mutation C400S, mutations V78A,F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V,L353V, I366V, T438S, and E442K, and at least one or more mutations atpositions A328 and/or L437 relative to the amino acid sequence set forthin SEQ ID NO:1 (SEQ ID NO: 54). In some instances, the cytochrome P450BM3 enzyme variant comprises an axial ligand mutation C400S andmutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,T268A, A290V, A328V, L353V, I366V, L437V, T438S, and E442K relative tothe amino acid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 51).

In some embodiments, the cytochrome P450 BM3 enzyme variant produces aplurality of aziridination products with a % ee_(S) of at least about75%. In some instances, the enzyme variant has a higher total turnovernumber (TTN) compared to the wild-type sequence. In some instances, theenzyme variant has a TTN greater than about 100.

In another aspect, the present invention provides a cytochrome P450 BM3enzyme variant or fragment thereof that can aziridinate an olefinicsubstrate comprising an axial ligand mutation C400S, mutations L75A,V87A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q, E252G, T268A,A290V, L353V, I366V, and E442K, and a mutation at position I263 relativeto the amino acid sequence set forth in SEQ ID NO: 1. In some instances,the enzyme variant comprises an axial ligand mutation C400S andmutations L75A, V78A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q,E252G, I263F, T268A, A290V, L353V, I366V, and E442K relative to theamino acid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 53).

In some embodiments, the cytochrome P450 BM3 enzyme variant produces aplurality of aziridination products with a % ee_(R) of at least about75%. In some instances, the enzyme variant has a higher total turnovernumber (TTN) compared to the wild-type sequence. In some instances, theenzyme variant has a TTN greater than about 100.

In certain aspects, the present invention provides a method forproducing an aziridination product, the method comprising:

-   -   (a) providing an olefinic substrate, a nitrene precursor, and a        heme enzyme; and    -   (b) admixing the components of step (a) in a reaction for a time        sufficient to produce an aziridination product.

In some embodiments, the method produces a plurality of aziridinationproducts. In some instances, the plurality of aziridination products hasa % ee_(S) of from about −90% to about 90%. In certain instances, theplurality of aziridination products has a % ee_(S) of from about −81% toabout 81%. In some instances, the plurality of aziridination productshas a Z:E ratio of from 1:99 to 99:1. In some instances, theaziridination reaction is at least 30% to at least 90%diastereoselective.

In some embodiments, the aziridination product is a compound accordingto Formula III:

wherein R^(1a), R^(1b), R³, and R³ can be as defined above in FormulaIII.

In certain other embodiments, the aziridination product is a compoundaccording to Formula IIIa:

wherein R^(1a), R^(1b), R³, and R³ can be as defined above in FormulaIII.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a heme enzyme intermolecular nitrogen-atom transfer inaccordance with an embodiment of the invention.

FIGS. 2A-B show HPLC 220 nm chromatograms of controls: FIG.2A—Co-injection of 4-methoxystyrene (Sigma Aldrich) and anN-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine synthetic standard,confirmed by NMR; FIG. 2B—Injection of theN-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine synthetic standardalone.

FIGS. 3A-D show HPLC 220 nm chromatograms of P411-enzymatic reactionswith 4-methoxystyrene and tosyl azide as substrates analyzed atdifferent time points. Putative N-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine and amido-alcohol derivative((N-(2-hydroxy-2-(4-methoxyphenyl)ethyl)-4-methylbenzenesulfonamide, 2)are marked with arrows.

FIGS. 4A-D show HPLC 220 nm chromatograms of synthetic standard S1,synthesized as previously reported, in reaction conditions without P411catalyst at several time points. Putative aziridine and amido-alcoholare marked with arrows, as in FIGS. 3A-D.

FIG. 5 shows a comparison of P-I263F productivity in vitro (purifiedprotein) and in whole cells.

FIG. 6 shows initial rates of aziridination and azide reduction forengineered enzymes. Total turnover (TTN) values were determined usingthe same method as described for initial rates, with the exception thatreactions were allowed to proceed for 4 hours in the anaerobic chamber.

FIGS. 7A-C show data used to determine initial rates for enzymes (A)P-I263F, (B) P-I263F-A328V, and (C) P-I263F-A328V-L437V. Diamondsrepresent concentrations of tosyl sulfonamide 7 and triangles representconcentrations of aziridine 4 for all plots.

FIG. 8 shows activity and selectivity of P-I263F-A328V-L437V withincreased substrate loading. Reactions were performed with whole E. colicells expressing P-I263F-A328V-L437V as described in the generalmethods, except substrate loading was increased to final concentrationsof 7.5 mM tosyl azide and 15 mM olefin.

FIGS. 9A-B are an exemplary demonstration of how absolutestereochemistry can be defined for the products herein. The assignmentof absolute stereochemistry of enzymatically produced aziridine 6 isassigned using chiral HPLC (Chiracel OJ, 30% isopropanol: 70% n-hexane,210 nm). Top: Racemic synthetic aziridine 6, t_(R)=16.7 min and 21.0min; Bottom: P-I263F-A328V-L437V produced aziridine 6, t_(R)=16.8 min.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, on the surprising discoverythat heme enzymes can be used to catalyze the conversion of olefinicbonds to aziridination products in the presence of nitrene precursors.FIG. 1 illustrates an exemplary reaction where styrene is converted toan aziridination product. In some aspects, cytochrome P450 BM3 enzymesand variants thereof were identified as having an unexpectedly efficientability to catalyze the formal transfer of nitrene equivalents fromnitrene precursors to various olefinic substrates, thereby makingaziridination products with high regioselectivity and/orstereoselectivity. In particular embodiments, the present inventors havediscovered that variants of P450 BM3 with at least one or more aminoacid mutations such as an axial ligand C400S, mutations V78A, F87V,P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V,I366V, T438S, and E442K, and at least one or more mutations at positionsA328 and/or L437 (SEQ ID NO: 54) can catalyze aziridination reactionsefficiently, displaying increased total turnover numbers (TTN) anddemonstrating highly regio- and enantioselective product formationcompared to wild-type enzymes.

Aziridination reactions can be performed by the heme enzymes describedherein in vitro or in vivo, where the heme enzyme is localized within awhole cell. In some embodiments, the heme enzyme described herein cancatalyze the aziridination reaction in vivo, providing over 500 totalturnovers with high stereoselectivity and yield.

The disclosure herein highlights the utility of enzymes in catalyzingnew types of reactions. The ability to genetically encode catalysts forformal nitrene transfers opens up new biosynthetic pathways to aminesand expands the scope of transformations accessible to biocatalysis.

II. Definitions

The following definitions and abbreviations are to be used for theinterpretation of the invention. The term “invention” or “presentinvention” as used herein is a non-limiting term and is not intended torefer to any single embodiment but encompasses all possible embodiments.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a species” includesa plurality of such species and reference to “the enzyme” includesreference to one or more enzymes and equivalents thereof, and so forth.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having, “contains,” “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Acomposition, mixture, process, method, article, or apparatus thatcomprises a list of elements is not necessarily limited to only thoseelements but may include other elements not expressly listed or inherentto such composition, mixture, process, method, article, or apparatus.Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “or.”

“About” and “around,” as used herein to modify a numerical value,indicate a defined range around that value. If “X” were the value,“about X” or “around X” would generally indicate a value from 0.95X to1.05X. Any reference to “about X” or “around X” specifically indicatesat least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X,1.03X, 1.04X, and 1.05X. Thus, “about X” and “around X” are intended toteach and provide written description support for a claim limitation of,e.g., “0.98X.” When the quantity “X” only includes whole-integer values(e.g., “X carbons”), “about X” or “around X” indicates from (X−1) to(X+1). In such cases, “about X” or “around X” specifically indicates atleast the values X, X−1, and X+1.

The term “aziridination (enzyme) catalyst” or “enzyme with aziridinationactivity” refers to any and all chemical processes catalyzed by enzymes,by which substrates containing at least one carbon-carbon double bondcan be converted into an aziridination product by using nitreneprecursors.

The terms “engineered heme enzyme” and “heme enzyme variant” include anyheme enzyme comprising at least one amino acid mutation with respect towild-type and also include any chimeric protein comprising recombinedsequences or blocks of amino acids from two, three, or more differentheme enzymes.

The terms “engineered cytochrome P450” and “cytochrome P450 variant”include any cytochrome P450 enzyme comprising at least one amino acidmutation with respect to wild-type and also include any chimeric proteincomprising recombined sequences or blocks of amino acids from two,three, or more different cytochrome P450 enzymes.

The term “whole cell catalyst” includes microbial cells expressing atleast one engineered heme enzyme, wherein the whole cell catalystdisplays aziridination activity.

As used herein, the terms “porphyrin” and “metal-substituted porphyrins”include any porphyrin that can be bound by a heme enzyme or variantthereof. In particular embodiments, these porphyrins may contain metalsincluding, but not limited to, Fe, Mn, Co, Cu, Rh, and Ru.

The term “heme” or “heme domain” as used herein refers to an amino acidsequence within an enzyme, which is capable of binding aniron-complexing structure such as a porphyrin. Compounds of iron aretypically complexed in a porphyrin (tetrapyrrole) ring that may differin side chain composition. Heme groups can be the prosthetic groups ofcytochromes and are found in most oxygen carrier proteins. Exemplaryheme domains include that of P450 BM3 as well as truncated or mutatedversions of these that retain the capability to bind the iron-complexingstructure. A skilled person can identify the heme domain of a specificprotein using methods known in the art.

The terms “nitrene equivalent” and “nitrene precursor” include moleculesthat can be decomposed in the presence of metal (or enzyme) catalysts tostructures that contain at least one nitrogen with only 5 valence shellelectrons and that can be transferred to C═C bonds to form aziridines.Nitrene precursors of the present invention include, but are not limitedto, sulfonyl azides, carbonyl azides, aryl azides, azidoformates,phosphoryl azides, azide phosphonates, iminoiodanes, or haloaminederivatives.

The terms “nitrene transfer” and “formal nitrene transfer” as usedherein include any chemical transformation where nitrene equivalents areadded to C═C bonds.

As used herein, the terms “microbial,” “microbial organism” and“microorganism” include any organism that exists as a microscopic cellthat is included within the domains of archaea, bacteria or eukarya.Therefore, the term is intended to encompass prokaryotic or eukaryoticcells or organisms having a microscopic size and includes bacteria,archaea and eubacteria of all species as well as eukaryoticmicroorganisms such as yeast and fungi. Also included are cell culturesof any species that can be cultured for the production of a chemical.

As used herein, the term “non-naturally occurring”, when used inreference to a microbial organism or enzyme activity of the invention,is intended to mean that the microbial organism or enzyme has at leastone genetic alteration not normally found in a naturally occurringstrain of the referenced species, including wild-type strains of thereferenced species. Genetic alterations include, for example,modifications introducing expressible nucleic acids encoding metabolicpolypeptides, other nucleic acid additions, nucleic acid deletionsand/or other functional disruption of the microbial organism's geneticmaterial. Such modifications include, for example, coding regions andfunctional fragments thereof, for heterologous, homologous or bothheterologous and homologous polypeptides for the referenced species.Additional modifications include, for example, non-coding regulatoryregions in which the modifications alter expression of a gene or operon.Exemplary non-naturally occurring microbial organism or enzyme activityincludes the aziridination activity described below.

As used herein, the term “anaerobic”, when used in reference to areaction, culture or growth condition, is intended to mean that theconcentration of oxygen is less than about 25 μM, preferably less thanabout 5 μM, and even more preferably less than 1 μM. The term is alsointended to include sealed chambers of liquid or solid medium maintainedwith an atmosphere of less than about 1% oxygen. Preferably, anaerobicconditions are achieved by sparging a reaction mixture with an inert gassuch as nitrogen or argon.

As used herein, the term “exogenous” is intended to mean that thereferenced molecule or the referenced activity is introduced into thehost microbial organism. The term as it is used in reference toexpression of an encoding nucleic acid refers to the introduction of theencoding nucleic acid in an expressible form into the microbialorganism. When used in reference to a biosynthetic activity, the termrefers to an activity that is introduced into the host referenceorganism.

The term “heterologous” as used herein with reference to molecules, andin particular enzymes and polynucleotides, indicates molecules that areexpressed in an organism other than the organism from which theyoriginated or are found in nature, independently of the level ofexpression that can be lower, equal or higher than the level ofexpression of the molecule in the native microorganism.

On the other hand, the term “native” or “endogenous” as used herein withreference to molecules, and in particular enzymes and polynucleotides,indicates molecules that are expressed in the organism in which theyoriginated or are found in nature, independently of the level ofexpression that can be lower equal or higher than the level ofexpression of the molecule in the native microorganism. It is understoodthat expression of native enzymes or polynucleotides may be modified inrecombinant microorganisms.

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In particular embodiments,the homology between two proteins is indicative of its shared ancestry,related by evolution.

The terms “analog” and “analogous” include nucleic acid or proteinsequences or protein structures that are related to one another infunction only and are not from common descent or do not share a commonancestral sequence. Analogs may differ in sequence but may share asimilar structure, due to convergent evolution. For example, two enzymesare analogs or analogous if the enzymes catalyze the same reaction ofconversion of a substrate to a product, are unrelated in sequence, andirrespective of whether the two enzymes are related in structure.

As used herein, the term “electron withdrawing group” refers to an atomor substituent that has an ability to acquire electron density from anolefin or other atoms or substituents. An “electron withdrawing group”is capable of withdrawing electron density relative to that of hydrogenif the hydrogen atom occupied the same position on the molecule. Theterm “electron withdrawing group” is well understood by those of skillin the art and is discussed, for example, in Advanced Organic Chemistryby J. March, John Wiley & Sons, New York, N.Y., (1985). Examples ofelectron withdrawing groups include, but are not limited to, halo (e.g.,fluorine, chlorine, bromine, iodine), nitro, carboxy, amido, acyl,cyano, aryl, heteroaryl, —OC(A)₃, —C(A)₃, —C(A)₂-O-C(A′)₃, —(CO)-Q,—SO₂—C(A)₃, —SO₂-aryl, —C(NQ)Q, —CH═C(Q)₂, and —C≡C-Q; in which each Aand A′ is independently H, halo, —CN, —NO₂, —OH, or C₁₋₄ alkyloptionally substituted with 1-3 halo, —OH, or NO₂; and Q is selectedfrom H, —OH, and alkyl optionally substituted with 1-3 halo, —OH,—O-alkyl, or —O-cycloalkyl.

The term “alkyl” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. Alkyl can includeany number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋ ₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 20 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated, and linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkylene can be linked to the same atom ordifferent atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is 1, 2, 3,4, 5 or 6. Representative alkylene groups include, but are not limitedto, methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, pentylene and hexylene. Alkylene groups can be substitutedor unsubstituted.

The term “alkoxy” refers to an alkyl group having an oxygen atom thatconnects the alkyl group to the point of attachment: alkyl-O—. As foralkyl group, alkoxy groups can have any suitable number of carbon atoms,such as C₁₋₆. Alkoxy groups include, for example, methoxy, ethoxy,propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy,tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be furthersubstituted with a variety of substituents described within. Alkoxygroups can be substituted or unsubstituted.

As used herein, the terms “halo” and “halogen” refer to fluorine,chlorine, bromine and iodine.

The term “heteroalkyl” refers to an alkyl group of any suitable lengthand having from 1 to 3 heteroatoms such as N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized, such as, but not limitedto, —SO— and —SO₂—. For example, heteroalkyl can include ethers,thioethers and alkyl-amines. The heteroatom portion of the heteroalkylcan be the connecting atom, or be inserted between two carbon atoms.

The term “aryl” refers to an aromatic ring system having any suitablenumber of ring atoms and any suitable number of rings. Aryl groups caninclude any suitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6to 14 ring members. Aryl groups can be monocyclic, fused to formbicyclic or tricyclic groups, or linked by a bond to form a biarylgroup. Representative aryl groups include phenyl, naphthyl and biphenyl.Other aryl groups include benzyl, having a methylene linking group. Somearyl groups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl. Aryl groups can be substituted or unsubstituted.

The term “heteroaryl” refers to a monocyclic or fused bicyclic ortricyclic aromatic ring assembly containing 5 to 16 ring atoms, wherefrom 1 to 5 of the ring atoms are a heteroatom such as N, O or S.Additional heteroatoms can also be useful, including, but not limitedto, B, Al, Si and P. The heteroatoms can also be oxidized, such as, butnot limited to, S═O and SO₂ (two double bonded oxygens). Heteroarylgroups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to12 ring members. Any suitable number of heteroatoms can be included inthe heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groupscan have from 5 to 8 ring members and from 1 to 4 heteroatoms, or from 5to 8 ring members and from 1 to 3 heteroatoms, or from 5 to 6 ringmembers and from 1 to 4 heteroatoms, or from 5 to 6 ring members andfrom 1 to 3 heteroatoms. The heteroaryl group can include groups such aspyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine,pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers),thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Theheteroaryl groups can also be fused to aromatic ring systems, such as aphenyl ring, to form members including, but not limited to,benzopyrroles such as indole and isoindole, benzopyridines such asquinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine(quinazoline), benzopyridazines such as phthalazine and cinnoline,benzothiophene, and benzofuran. Other heteroaryl groups includeheteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groupscan be substituted or unsubstituted. Heteroaryl groups can be linked viaany position on the ring.

The term “cycloalkyl” refers to a saturated or partially unsaturated,monocyclic, fused bicyclic or bridged polycyclic ring assemblycontaining from 3 to 12 ring atoms, or the number of atoms indicated.Cycloalkyl can include any number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆,C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈, C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturatedmonocyclic cycloalkyl rings include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclicand polycyclic cycloalkyl rings include, for example, norbornane,[2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkylgroups can also be partially unsaturated, having one or more double ortriple bonds in the ring. Representative cycloalkyl groups that arepartially unsaturated include, but are not limited to, cyclobutene,cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers),cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-and 1,5-isomers), norbornene, and norbornadiene. When cycloalkyl is asaturated monocyclic C₃₋₈ cycloalkyl, exemplary groups include, but arenot limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclicC₃₋₆ cycloalkyl, exemplary groups include, but are not limited tocyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groupscan be substituted or unsubstituted.

The term “iodinane” refers to the chemical substituent

where the ‘wavy line’ represents the point of attachment to theremainder of the molecule.

Any compound or formula disclosed herein that does not define thechirality of a chiral carbon can be a racemic mixture or may possess anenantiomeric excess of R or S isomers. For example, compoundsrepresented by Formula III, shown below, may possess, 0, 1, or 2 chiralcarbons depending on the identities of R^(1a), R^(1b), and R². Eachchiral center may be racemic or may be of a particular enantiomericexcess. A person of skill in the art will also recognize that instancesof two chiral carbons within an single aziridine ring can produce twocis and two trans isomers.

III. Description of the Embodiments

In a first aspect, the invention provides a reaction mixture forproducing an aziridination product. The reaction mixture contains anolefinic substrate, a nitrene precursor, and a heme enzyme.

In other aspects, the present invention provides heme enzymes includingvariants thereof that are capable of carrying out the aziridinationreactions described herein. Expression vectors and host cells expressingthe heme enzymes are also provided by the present invention.

In still other aspects, the present invention provides methods forproducing an aziridination product. In certain aspects, the presentinvention provides a method for producing an aziridination product, themethod comprising:

-   -   (a) providing an olefinic substrate, a nitrene precursor, and a        heme enzyme; and    -   (b) admixing the components of step (a) in a reaction for a time        sufficient to produce an aziridination product.

The following sections provide a description of exemplary and preferredembodiments including heme enzymes, expression vectors, host cells,aziridination products such as, e.g., compounds comprising an aziridinefunctional group, starting materials such as, e.g., olefinic substratesand nitrene precursors, and characteristics and reaction conditions forthe in vitro and in vivo aziridination reactions described herein.

A. Heme Enzymes

The terms “heme enzyme” and “heme protein” are used herein to includeany member of a group of proteins containing heme as a prosthetic group.Non-limiting examples of heme enzymes include globins, cytochromes,oxidoreductases, any other protein containing a heme as a prostheticgroup, and combinations thereof. Heme-containing globins include, butare not limited to, hemoglobin, myoglobin, and combinations thereof.Heme-containing cytochromes include, but are not limited to, cytochromeP450, cytochrome b, cytochrome c1, cytochrome c, and combinationsthereof. Heme-containing oxidoreductases include, but are not limitedto, a catalase, an oxidase, an oxygenase, a haloperoxidase, aperoxidase, and combinations thereof.

In certain aspects, the present invention provides compositionscomprising one or more heme enzymes that catalyze the conversion ofolefinic substrates to aziridination products. In particularembodiments, the present invention provides heme enzyme variantscomprising at least one or more amino acid mutations therein thatcatalyze the formal transfer of a nitrene equivalent to an olefinicsubstrate, making aziridination products with high stereoselectivity. Inpreferred embodiments, the heme enzyme variants of the present inventionhave the ability to catalyze aziridination reactions efficiently,display increased total turnover numbers, and/or demonstrate highlyregio- and/or enantioselective product formation compared to thecorresponding wild-type enzymes.

In some embodiments, the enzyme is a heme enzyme or a variant thereof.In certain instances, the heme enzymes are metal-substituted hemeenzymes containing protoporphyrin IX or other porphyrin moleculescontaining metals other than iron, including, but not limited to,cobalt, rhodium, copper, ruthenium, and manganese, which are activeaziridination catalysts.

In some embodiments, the heme enzyme is a member of one of the enzymeclasses set forth in Table A. In other embodiments, the heme enzyme is avariant or homolog of a member of one of the enzyme classes set forth inTable A. In yet other embodiments, the heme enzyme comprises or consistsof the heme domain of a member of one of the enzyme classes set forth inTable A or a fragment thereof (e.g., a truncated heme domain) that iscapable of carrying out the aziridination reactions described herein.

TABLE A Heme enzymes identified by their enzyme classification number(EC number) and classification name. EC Number Name 1.1.2.3 L-lactatedehydrogenase 1.1.2.6 polyvinyl alcohol dehydrogenase (cytochrome)1.1.2.7 methanol dehydrogenase (cytochrome c) 1.1.5.5 alcoholdehydrogenase (quinone) 1.1.5.6 formate dehydrogenase-N: 1.1.9.1 alcoholdehydrogenase (azurin): 1.1.99.3 gluconate 2-dehydrogenase (acceptor)1.1.99.11 fructose 5-dehydrogenase 1.1.99.18 cellobiose dehydrogenase(acceptor) 1.1.99.20 alkan-1-ol dehydrogenase (acceptor) 1.2.1.70glutamyl-tRNA reductase 1.2.3.7 indole-3-acetaldehyde oxidase 1.2.99.3aldehyde dehydrogenase (pyrroloquinoline-quinone) 1.3.1.6 fumaratereductase (NADH): 1.3.5.1 succinate dehydrogenase (ubiquinone) 1.3.5.4fumarate reductase (menaquinone) 1.3.99.1 succinate dehydrogenase1.4.9.1 methylamine dehydrogenase (amicyanin) 1.4.9.2. aralkylaminedehydrogenase (azurin) 1.5.1.20 methylenetetrahydrofolate reductase[NAD(P)H] 1.5.99.6 spermidine dehydrogenase 1.6.3.1 NAD(P)H oxidase1.7.1.1 nitrate reductase (NADH) 1.7.1.2 Nitrate reductase [NAD(P)H]1.7.1.3 nitrate reductase (NADPH) 1.7.1.4 nitrite reductase [NAD(P)H]1.7.1.14 nitric oxide reductase ]NAD(P), nitrous oxide-forming] 1.7.2.1nitrite reductase (NO-forming) 1.7.2.2 nitrite reductase (cytochrome;ammonia-forming) 1.7.2.3 trimethylamine-N-oxide reductase (cytochrome c)1.7.2.5 nitric oxide reductase (cytochrome c) 1.7.2.6 hydroxylaminedehydrogenase 1.7.3.6 hydroxylamine oxidase (cytochrome) 1.7.5.1 nitratereductase (quinone) 1.7.5.2 nitric oxide reductase (menaquinol) 1.7.6.1nitrite dismutase 1.7.7.1 ferredoxin-nitrite reductase 1.7.7.2ferredoxin-nitrate reductase 1.7.99.4 nitrate reductase 1.7.99.8hydrazine oxidoreductase 1.8.1.2 sulfite reductase (NADPH) 1.8.2.1sulfite dehydrogenase 1.8.2.2 thiosulfate dehydrogenase 1.8.2.3sulfide-cytochrome-c reductase (flavocytochrome c) 1.8.2.4 dimethylsulfide:cytochrome c2 reductase 1.8.3.1 sulfite oxidase 1.8.7.1 sulfitereductase (ferredoxin) 1.8.98.1 CoB-CoM heterodisulfide reductase1.8.99.1 sulfite reductase 1.8.99.2 adenylyl-sulfate reductase 1.8.99.3hydrogensulfite reductase 1.9.3.1 cytochrome-c oxidase 1.9.6.1 nitratereductase (cytochrome) 1.10.2.2 ubiquinol-cytochrome-c reductase1.10.3.1 catechol oxidase 1.10.3.B1 caldariellaquinol oxidase(H+-transporting) 1.10.3.3 L-ascothate oxidase 1.10.3.9 photosystem II1.10.3.10 ubiquinol oxidase (H+-transporting) 1.10.3.11 ubiquinoloxidase 1.10.3.12 menaquinol oxidase (H+-transporting) 1.10.9.1plastoquinol-plastocyanin reductase 1.11.1.5 cytochrome-c peroxidase1.11.1.6 catalase 1.11.1.7 peroxidase 1.11.1.B2 chloride peroxidase(vanadium-containing) 1.11.1.B7 bromide peroxidase (heme-containing)1.11.1.8 iodide peroxidase 1.11.1.10 chloride peroxidase 1.11.1.11L-ascothate peroxidase 1.11.1.13 manganese peroxidase 1.11.1.14 ligninperoxidase 1.11.1.16 versatile peroxidase 1.11.1.19 dye decolorizingperoxidase 1.11.1.21 catalase-peroxidase 1.11.2.1 unspecificperoxygenase 1.11.2.2 myeloperoxidase 1.11.2.3 plant seed peroxygenase1.11.2.4 fatty-acid peroxygenase 1.12.2.1 cytochrome-c3 hydrogenase1.12.5.1 hydrogen:quinone oxidoreductase 1.12.99.6 hydrogenase(acceptor) 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase 1.13.11.11tryptophan 2,3-dioxygenase 1.13.11.49 chlorite O2-lyase 1.13.11.50acetylacetone-cleaving enzyme 1.13.11.52 indoleamine 2,3-dioxygenase1.13.11.60 linoleate 8R-lipoxygenase 1.13.99.3 tryptophan 21-dioxygenase1.14.11.9 flavanone 3-dioxygenase 1.14.12.17 nitric oxide dioxygenase1.14.13.39 nitric-oxide synthase (NADPH dependent) 1.14.13.17cholesterol 7alpha-monooxygenase 1.14.13.41 tyrosine N-monooxygenase1.14.13.70 sterol 14alpha-demethylase 1.14.13.71 N-methylcoclaurine3′-monooxygenase 1.14.13.81 magnesium-protoporphyrin IX monomethyl ester(oxidative) cyclase 1.14.13.86 2-hydroxyisoflavanone synthase 1.14.13.98cholesterol 24-hydroxylase 1.14.13.119 5-epiaristolochene1,3-dihydroxylase 1.14.13.126 vitamin D3 24-hydroxylase 1.14.13.129beta-carotene 3-hydroxylase 1.14.13.141 cholest-4-en-3-one26-monooxygenase 1.14.13.142 3-ketosteroid 9alpha-monooxygenase1.14.13.151 linalool 8-monooxygenase 1.14.13.156 1,8-cineole2-endo-monooxygenase 1.14.13.159 vitamin D 25-hydroxylase 1.14.14.1unspecific monooxygenase 1.14.15.1 camphor 5-monooxygenase 1.14.15.6cholesterol monooxygenase (side-chain-cleaving) 1.14.15.8 steroid15beta-monooxygenase 1.14.15.9 spheroidene monooxygenase 1.14.18.1tyrosinase 1.14.19.1 stearoyl-CoA 9-desaturase 1.14.19.3 linoleoyl-CoAdesaturase 1.14.21.7 biflaviolin synthase 1.14.99.1prostaglandin-endoperoxide synthase 1.14.99.3 heme oxygenase 1.14.99.9steroid 17alpha-monooxygenase 1.14.99.10 steroid 21-monooxygenase1.14.99.15 4-methoxybenzoate monooxygenase (O-demethylating) 1.14.99.45carotene epsilon-monooxygenase 1.16.5.1 ascorbate ferrireductase(transmembrane) 1.16.9.1 iron:rusticyanin reductase 1.17.1.4 xanthinedehydrogenase 1.17.2.2 lupanine 17-hydroxylase (cytochrome c) 1.17.99.14-methylphenol dehydrogenase (hydroxylating) 1.17.99.2 ethylbenzenehydroxylase 1.97.1.1 chlorate reductase 1.97.1.9 selenate reductase2.7.7.65 diguanylate cyclase 2.7.13.3 histidine kinase 3.1.4.52cyclic-guanylate-specific phosphodiesterase 4.2.1.B9 colneleicacid/etheroleic acid synthase 4.2.1.22 Cystathionine beta-synthase4.2.1.92 hydroperoxide dehydratase 4.2.1.212 colneleate synthase4.3.1.26 chromopyrrolate synthase 4.6.1.2 guanylate cyclase 4.99.1.3sirohydrochlorin cobaltochelatase 4.99.1.5 aliphatic aldoximedehydratase 4.99.1.7 phenylacetaldoxime dehydratase 5.3.99.3prostaglandin-E synthase 5.3.99.4 prostaglandin-I synthase 5.3.99.5Thromboxane-A synthase 5.4.4.5 9,12-octadecadienoate 8-hydroperoxide8R-isomerase 5.4.4.6 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase6.6.1.2 cobaltochelatase

In some embodiments, the heme enzyme is a variant or a fragment thereof(e.g., a truncated variant containing the heme domain) comprising atleast one mutation such as, e.g., a mutation at the axial position ofthe heme coordination site. In some instances, the mutation is asubstitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu,Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Valat the axial position. In certain instances, the mutation is asubstitution of Cys with any other amino acid such as Ser at the axialposition.

In certain embodiments, the in vitro methods for producing anaziridination product comprise providing a heme enzyme, variant, orhomolog thereof with a reducing agent such as NADPH or a dithionite salt(e.g., Na₂S₂O₄). In certain other embodiments, the in vivo methods forproducing an aziridination product comprise providing whole cells suchas E. coli cells expressing a heme enzyme, variant, or homolog thereof.

In some embodiments, the heme enzyme, variant, or homolog thereof isrecombinantly expressed and optionally isolated and/or purified forcarrying out the in vitro aziridination reactions of the presentinvention. In other embodiments, the heme enzyme, variant, or homologthereof is expressed in whole cells such as E. coli cells, and thesecells are used for carrying out the in vivo aziridination reactions ofthe present invention.

In certain embodiments, the heme enzyme, variant, or homolog thereofcomprises or consists of the same number of amino acid residues as thewild-type enzyme (e.g., a full-length polypeptide). In some instances,the heme enzyme, variant, or homolog thereof comprises or consists of anamino acid sequence without the start methionine (e.g., P450 BM3 aminoacid sequence set forth in SEQ ID NO:1). In other embodiments, the hemeenzyme comprises or consists of a heme domain fused to a reductasedomain. In yet other embodiments, the heme enzyme does not contain areductase domain, e.g., the heme enzyme contains a heme domain only or afragment thereof such as a truncated heme domain.

In some embodiments, the heme enzyme, variant, or homolog thereof has anenhanced nitrene insertion activity and/or nitrene transfer activity ofabout 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold compared to thecorresponding wild-type heme enzyme.

In some embodiments, the heme enzyme, variant, or homolog thereof has aresting state reduction potential higher than that of NADH or NADPH.

In particular embodiments, the heme enzyme comprises a cyctochrome P450enzyme. Cytochrome P450 enzymes constitute a large superfamily ofheme-thiolate proteins involved in the metabolism of a wide variety ofboth exogenous and endogenous compounds. Usually, they act as theterminal oxidase in multicomponent electron transfer chains, such asP450-containing monooxygenase systems. Members of the cytochrome P450enzyme family catalyze myriad oxidative transformations, including,e.g., hydroxylation, epoxidation, oxidative ring coupling, heteroatomrelease, and heteroatom oxygenation (E. M. Isin et al., Biochim.Biophys. Acta 1770, 314 (2007)). The active site of these enzymescontains an FeIII-protoporphyrin IX cofactor (heme) ligated proximallyby a conserved cysteine thiolate (M. T. Green, Current Opinion inChemical Biology 13, 84 (2009)).

One skilled in the art will appreciate that the cytochrome P450 enzymesuperfamily has been compiled in various databases, including, but notlimited to, the P450 homepage (available athttp://drnelson.uthsc.edu/CytochromeP450.html; see also, D. R. Nelson,Hum. Genomics 4, 59 (2009)), the cytochrome P450 enzyme engineeringdatabase (available athttp://www.cyped.uni-stuttgart.de/cgi-bin/CYPED5/index.pl; see also, D.Sirim et al., BMC Biochem 10, 27 (2009)), and the SuperCyp database(available at http://bioinformatics.charite.de/supercyp/; see also, S.Preissner et al., Nucleic Acids Res. 38, D237 (2010)), the disclosuresof which are incorporated herein by reference in their entirety for allpurposes.

In certain embodiments, the cytochrome P450 enzymes of the invention aremembers of one of the classes shown in Table B (see,http://www.icgeb.org/˜p450srv/P450enzymes.html, the disclosure of whichis incorporated herein by reference in its entirety for all purposes).

TABLE B Cytochrome P450 enzymes classified by their EC number,recommended name, and family/gene name. EC Recommended name Family/gene1.3.3.9 secologanin synthase CYP72A1 1.14.13.11 trans-cinnamate4-monooxygenase CYP73 1.14.13.12 benzoate 4-monooxygenase CYP531.14.13.13 calcidiol 1-monooxygenase CYP27 1.14.13.15 cholestanetriol26-monooxygenase CYP27 1.14.13.17 cholesterol 7x-monooxygenase CYP71.14.13.21 flavonoid 3′-monooxygenase CYP75 1.14.13.283,9-dihydroxypterocarpan 6a-monooxygenase CYP93A1 1.14.13.30leukotriene-B₄ 20-monooxygenase CYP4F 1.14.13.37methyltetrahydroprotoberberine 14-monooxygenase CYP93A1 1.14.13.41tyrosine N-monooxygenase CYP79 1.14.13.42 hydroxyphenylacetonitrile2-monooxygenase — 1.14.13.47 (-)-limonene 3-monooxygenase — 1.14.13.48(-)-limonene 6-monooxygenase — 1.14.13.49 (-)-limonene 7-monooxygenase —1.14.13.52 isoflavone 3′-hydroxylase — 1.14.13.53 isoflavone2′-hydroxylase — 1.14.13.55 protopine 6-monooxygenase — 1.14.13.56dihydrosanguinarine 10-monooxygenase — 1.14.13.57 dihydrochelirubine12-monooxygenase — 1.14.13.60 27-hydroxycholesterol 7x-monooxygenase —1.14.13.70 sterol 14-demethylase CYP51 1.14.13.71 N-methylcoclaurine3′-monooxygenase CYP80B1 1.14.13.73 tabersonine 16-hydroxylase CYP71D121.14.13.74 7-deoxyloganin 7-hydroxylase — 1.14.13.75 vinorinehydroxylase — 1.14.13.76 taxane 10β-hydroxylase CYP725A1 1.14.13.77taxane 13α-hydroxylase CYP725A2 1.14.13.78 ent-kaurene oxidase CYP7011.14.13.79 ent-kaurenoic acid oxidase CYP88A 1.14.14.1 unspecificmonooxygenase multiple 1.14.15.1 camphor 5-monooxygenase CYP1011.14.15.3 alkane 1-monooxygenase CYP4A 1.14.15.4 steroid11β-monooxygenase CYP11B 1.14.15.5 corticosterone 18-monooxygenaseCYP11B 1.14.15.6 cholesterol monooxygenase (side-chain-cleaving) CYP11A1.14.21.1 (S)-stylopine synthase — 1.14.21.2 (S)-cheilanthifolinesynthase — 1.14.21.3 berbamunine synthase CYP80 1.14.21.4 salutaridinesynthase — 1.14.21.5 (S)-canadine synthase — 1.14.99.9 steroid17x-monooxygenase CYP17 1.14.99.10 steroid 21-monooxygenase CYP211.14.99.22 ecdysone 20-monooxygenase — 1.14.99.28 linalool8-monooxygenase CYP111 4.2.1.92 hydroperoxide dehydratase CYP74 5.3.99.4prostaglandin-I synthase CYP8 5.3.99.5 thromboxane-A synthase CYP5

Table C below lists additional cyctochrome P450 enzymes that aresuitable for use in the aziridination reactions of the presentinvention. The accession numbers in Table C are incorporated herein byreference in their entirety for all purposes. The cytochrome P450 geneand/or protein sequences disclosed in the following patent documents arehereby incorporated by reference in their entirety for all purposes: WO2013/076258; CN 103160521; CN 103223219; KR 2013081394; JP 5222410; WO2013/073775; WO 2013/054890; WO 2013/048898; WO 2013/031975; WO2013/064411; U.S. Pat. No. 8,361,769; WO 2012/150326, CN 102747053; CN102747052; JP 2012170409; WO 2013/115484; CN 103223219; KR 2013081394;CN 103194461; JP 5222410; WO 2013/086499; WO 2013/076258; WO2013/073775; WO 2013/064411; WO 2013/054890; WO 2013/031975; U.S. Pat.No. 8,361,769; WO 2012/156976; WO 2012/150326; CN 102747053; CN102747052; US 20120258938; JP 2012170409; CN 102399796; JP 2012055274;WO 2012/029914; WO 2012/028709; WO 2011/154523; JP 2011234631; WO2011/121456; EP 2366782; WO 2011/105241; CN 102154234; WO 2011/093185;WO 2011/093187; WO 2011/093186; DE 102010000168; CN 102115757; CN102093984; CN 102080069; JP 2011103864; WO 2011/042143; WO 2011/038313;JP 2011055721; WO 2011/025203; JP 2011024534; WO 2011/008231; WO2011/008232; WO 2011/005786; IN 2009DE01216; DE 102009025996; WO2010/134096; JP 2010233523; JP 2010220609; WO 2010/095721; WO2010/064764; US 20100136595; JP 2010051174; WO 2010/024437; WO2010/011882; WO 2009/108388; US 20090209010; US 20090124515; WO2009/041470; KR 2009028942; WO 2009/039487; WO 2009/020231; JP2009005687; CN 101333520; CN 101333521; US 20080248545; JP 2008237110;CN 101275141; WO 2008/118545; WO 2008/115844; CN 101255408; CN101250506; CN 101250505; WO 2008/098198; WO 2008/096695; WO 2008/071673;WO 2008/073498; WO 2008/065370; WO 2008/067070; JP 2008127301; JP2008054644; KR 794395; EP 1881066; WO 2007/147827; CN 101078014; JP2007300852; WO 2007/048235; WO 2007/044688; WO 2007/032540; CN 1900286;CN 1900285; JP 2006340611; WO 2006/126723; KR 2006029792; KR 2006029795;WO 2006/105082; WO 2006/076094; US 2006/0156430; WO 2006/065126; JP2006129836; CN 1746293; WO 2006/029398; JP 2006034215; JP 2006034214; WO2006/009334; WO 2005/111216; WO 2005/080572; US 2005/0150002; WO2005/061699; WO 2005/052152; WO 2005/038033; WO 2005/038018; WO2005/030944; JP 2005065618; WO 2005/017106; WO 2005/017105; US20050037411; WO 2005/010166; JP 2005021106; JP 2005021104; JP2005021105; WO 2004/113527; CN 1472323; JP 2004261121; WO 2004/013339;WO 2004/011648; DE 10234126; WO 2004/003190; WO 2003/087381; WO2003/078577; US 20030170627; US 20030166176; US 20030150025; WO2003/057830; WO 2003/052050; CN 1358756; US 20030092658; US 20030078404;US 20030066103; WO 2003/014341; US 20030022334; WO 2003/008563; EP1270722; US 20020187538; WO 2002/092801; WO 2002/088341; US 20020160950;WO 2002/083868; US 20020142379; WO 2002/072758; WO 2002/064765; US20020076777; US 20020076774; US 20020076774; WO 2002/046386; WO2002/044213; US 20020061566; CN 1315335; WO 2002/034922; WO 2002/033057;WO 2002/029018; WO 2002/018558; JP 2002058490; US 20020022254; WO2002/008269; WO 2001/098461; WO 2001/081585; WO 2001/051622; WO2001/034780; CN 1271005; WO 2001/011071; WO 2001/007630; WO 2001/007574;WO 2000/078973; U.S. Pat. No. 6,130,077; JP 2000152788; WO 2000/031273;WO 2000/020566; WO 2000/000585; DE 19826821; JP 11235174; U.S. Pat. No.5,939,318; WO 99/19493; WO 99/18224; U.S. Pat. No. 5,886,157; WO99/08812; U.S. Pat. No. 5,869,283; JP 10262665; WO 98/40470; EP 776974;DE 19507546; GB 2294692; U.S. Pat. No. 5,516,674; JP 07147975; WO94/29434; JP 06205685; JP 05292959; JP 04144680; DD 298820; EP 477961;SU 1693043; JP 01047375; EP 281245; JP 62104583; JP 63044888; JP62236485; JP 62104582; and JP 62019084.

TABLE C Additional cytochrome P450 enzymes of the present invention.Species Cyp No. Accession No. SEQ ID NO Bacillus megaterium 102A1AAA87602 1 Bacillus megaterium 102A1 ADA57069 2 Bacillus megaterium102A1 ADA57068 3 Bacillus megaterium 102A1 ADA57062 4 Bacillusmegaterium 102A1 ADA57061 5 Bacillus megaterium 102A1 ADA57059 6Bacillus megaterium 102A1 ADA57058 7 Bacillus megaterium 102A1 ADA570558 Bacillus megaterium 102A1 ACZ37122 9 Bacillus megaterium 102A1ADA57057 10 Bacillus megaterium 102A1 ADA57056 11 Mycobacterium sp.HXN-1500 153A6 CAH04396 12 Tetrahymena thermophile 5013C2 ABY59989 13Nonomuraea dietziae AGE14547.1 14 Homo sapiens 2R1 NP_078790 15 Maccamulatta 2R1 NP_001180887.1 16 Canis familiaris 2R1 XP_854533 17 Musmusculus 2R1 AAI08963 18 Bacillus halodurans C-125 152A6 NP_242623 19Streptomyces parvus aryC AFM80022 20 Pseudomonas putida 101A1 P00183 21Homo sapiens 2D7 AAO49806 22 Rattus norvegicus C27 AAB02287 23Oryctolagus cuniculus 2B4 AAA65840 24 Bacillus subtilis 102A2 O08394 25Bacillus subtilis 102A3 O08336 26 B. megaterium DSM 32 102A1 P14779 27B. cereus ATCC14579 102A5 AAP10153 28 B. licheniformis ATTC1458 102A7 YP079990 29 B. thuringiensis serovar konkukian X YP 037304 30 str.97-27 R.metallidurans CH34 102E1 YP 585608 31 A. fumigatus Af293 505X EAL9266032 A. nidulans FGSC A4 505A8 EAA58234 33 A. oryzae ATCC42149 505A3Q2U4F1 34 A. oryzae ATCC42149 X Q2UNA2 35 F. oxysporum 505A1 Q9Y8G7 36G. moniliformis X AAG27132 37 G. zeae PH1 505A7 EAA67736 38 G. zeae PH1505C2 EAA77183 39 M. grisea 70-15 syn 505A5 XP 365223 40 N. crassa OR74A 505A2 XP 961848 41 Oryza sativa* 97A Oryza sativa* 97B Oryza sativa97C ABB47954 42 The start methionine (“M”) may be present or absent fromthese sequences. *See, M.Z. Lv et al., Plant Cell Physiol.,53(6):987-1002 (2012).

In certain embodiments, the present invention provides amino acidsubstitutions that efficiently remove monooxygenation chemistry fromcytochrome P450 enzymes. This system permits selective enzyme-drivenaziridination chemistry without competing side reactions mediated bynative P450 catalysis. The invention also provides P450-mediatedcatalysis that is competent for aziridination chemistry but not able tocarry out traditional P450-mediated monooxygenation reactions as‘orthogonal’ P450 catalysis and respective enzyme variants as‘orthogonal’ P450s. In some instances, orthogonal P450 variants comprisea single amino acid mutation at the axial position of the hemecoordination site (e.g., a C400S mutation in the P450 BM3 enzyme) thatalters the proximal heme coordination environment. Accordingly, thepresent invention also provides P450 variants that contain an axial hememutation in combination with one or more additional mutations describedherein to provide orthogonal P450 variants that show enricheddiastereoselective and/or enantioselective product distributions. Thepresent invention further provides a compatible reducing agent fororthogonal P450 aziridination catalysis that includes, but is notlimited to, NAD(P)H or sodium dithionite.

In certain instances, the cytochrome P450 BM3 enzyme comprises orconsists of the amino acid sequence set forth in SEQ ID NO:1. In certainother instances, the cytochrome P450 BM3 enzyme is a natural variantthereof as described, e.g., in J. Y. Kang et al., AMB Express 1:1(2011), wherein the natural variants are divergent in amino acidsequence from the wild-type cytochrome P450 BM3 enzyme sequence (SEQ IDNO:1) by up to about 5% (e.g., SEQ ID NOS:2-11).

In particular embodiments, the P450 BM3 enzyme variant comprises orconsists of the heme domain of the wild-type P450 BM3 enzyme sequence(e.g., amino acids 1-463 of SEQ ID NO: 1) and optionally at least onemutation as described herein. In other embodiments, the P450 BM3 enzymevariant comprises or consists of a fragment of the heme domain of thewild-type P450 BM3 enzyme sequence (SEQ ID NO: 1), wherein the fragmentis capable of carrying out the aziridination reactions of the presentinvention. In some instances, the fragment includes the heme axialligand and at least one, two, three, four, or five of the active siteresidues.

In other embodiments, the P450 BM3 enzyme variant comprises at least oneor more (e.g., at least two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or all fourteen) of the following aminoacid substitutions in SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V,S226R, H236Q, E252G, T268A, A290V, L353V, I366V, T438S, and E442K (SEQID NO: 55). In certain instances, the P450 BM3 enzyme variant comprisesa T268A mutation alone or in combination with one or more additionalmutations such as a C400X mutation (e.g., C400S) in SEQ ID NO:1 (SEQ IDNO: 56). In other instances, the P450 BM3 enzyme variant comprises allfourteen of these amino acid substitutions (i.e., V78A, F87V, P142S,T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, T438S,and E442K; “BM3-CIS T438S”) in combination with a C400X mutation (e.g.,C400S) in SEQ ID NO:1 (SEQ ID NO: 57). In some instances, the P450 BM3enzyme variant comprises or consists of the heme domain of the BM3-CIST438S enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO: 1comprising all fourteen of these amino acid substitutions (SEQ ID NO:55)).

In some embodiments, the P450 BM3 enzyme variant comprises the axialligand mutation C400S and substitutions to SEQ ID NO:1: V78A, F87V,P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, A328V,L353V, I366V, L437V, T438S, E442K (SEQ ID NO: 51). In anotherembodiment, the heme variant comprises the axial ligand mutation C400Sand the following amino acid substitutions: L75A, V78A, F87V, P142S,T175I, L181A, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V,I366V, E442K (SEQ ID NO: 53). In some embodiments, the heme enzymevariant is the P-I263F variant (see, Table D). In some embodiments, theheme enzyme variant is the P411_(BM3) H2-A-10 I263F (see, Table D).

Table D below provides non-limiting examples of cytochrome P450 BM3variants of the present invention. Each P450 BM3 variant comprises themutations relative to the wild-type P450 BM3 enzymes as shown.

TABLE D Mutations present in P450 BM3 variants used in the disclosure.P450_(BM3) variant Mutations relative to wild-type P450_(BM3) (SEQ IDNO: 1) P450_(BM3) none P450_(BM3)-T268A T268A P411_(BM3) C400SP411_(BM3)-T268A C400S, T268A P450_(BM3)-CIS V78A, F87V, P142S, T175I,A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, E442KP450_(BM3)-CIS-T438S CIS T438S P450_(BM3)-CIS-T438S C400H P450_(BM3)-CIST438S, C400H P450_(BM3)-CIS-T438S C400D P450_(BM3)-CIS T438S, C400DP450_(BM3)-CIS-T438S C400M P450_(BM3)-CIS T438S, C400M P411_(BM3)-CISP450_(BM3)-CIS C400S P411_(BM3)-CIS-T4385 P450_(BM3)-CIS C400S, T438SP411_(BM3)-CIS-T4385 I263F P450_(BM3)-CIS C400S, T438S, I263F (P-I263F)P-I263F-A328V P450_(BM3)-CIS C400S, T438S, I263F, A328VP-I263F-A328V-L437V P450_(BM3)-CIS C400S, T438S, I263F, A328V, L437VP411_(BM3)-CIS T438S I263F V87F P450_(BM3)-CIS C400S, T438S I263F, V87FP411_(BM3)-CIS T438S I263F A268T P450_(BM3)-CIS C400S, T438S I263F,A268T P411_(BM3)-CIS A268T T438S P450_(BM3)-CIS C400S, A268T, T438SP411_(BM3) H2-A-10 P450_(BM3)-CIS C400S, L75A, L181A P411_(BM3) H2-A-10I263F P450_(BM3)-CIS C400S, L75A, L181A, I263F P411_(BM3) H2-5-F10P450_(BM3)-CIS C400S, L75A, I263A, L437A P411_(BM3) H2-4-D4P450_(BM3)-CIS C400S, L75A, M177A, L181A, L437A

One skilled in the art will understand that any of the mutations listedin Table D can be introduced into any cytochrome P450 enzyme of interestby locating the segment of the DNA sequence in the correspondingcytochrome P450 gene which encodes the conserved amino acid residue asdescribed above for identifying the conserved cysteine residue in acytochrome P450 enzyme of interest that serves as the heme axial ligand.In certain instances, this DNA segment is identified through detailedmutagenesis studies in a conserved region of the protein (see, e.g.,Shimizu et al., Biochemistry 27, 4138-4141, 1988). In other instances,the conserved amino acid residue is identified through crystallographicstudy (see, e.g., Poulos et al., J. Mol. Biol 195:687-700, 1987). In yetother instances, protein sequence alignment algorithms can be used toidentify the conserved amino acid residue. For example, BLAST alignmentwith the P450 BM3 amino acid sequence as the query sequence can be usedto identify the heme axial ligand site and/or the equivalent T268residue in other cytochrome P450 enzymes.

In other aspects, the disclosure provides chimeric heme enzymes such as,e.g., chimeric P450 polypeptides comprised of recombined sequences fromP450 BM3 and at least two, or more distantly related P450 enzymes fromBacillus subtillis or variants. As a non-limiting example, site-directedrecombination of three bacterial cytochrome P450s can be performed withsequence crossover sites selected to minimize the number of disruptedcontacts within the protein structure. In some embodiments, sevencrossover sites can be chosen, resulting in eight sequence blocks. Oneskilled in the art will understand that the number of crossover sitescan be chosen to produce the desired number of sequence blocks, e.g., 1,2, 3, 4, 5, 6, 7, 8, or 9 crossover sites for 2, 3, 4, 5, 6, 7, 8, 9, or10 sequence blocks, respectively. In other embodiments, the numberingused for the chimeric P450 refers to the identity of the parent sequenceat each block. For example, “12312312” refers to a sequence containingblock 1 from P450 #1, block 2 from P450 #2, block 3 from P450 #3, block4 from P450 #1, block 5 from P450 #2, and so on. A chimeric libraryuseful for generating the chimeric heme enzymes of the invention can beconstructed as described in U.S. Pat. Publ. No. US-2012-0171693-A1 toArnold et al., the disclosure of which is incorporated herein for allpurposes.

As a non-limiting example, chimeric P450 proteins comprising recombinedsequences or blocks of amino acids from CYP102A1 (Accession No. J04832),CYP102A2 (Accession No. CAB12544), and CYP102A3 (Accession No. U93874)can be constructed. In certain instances, the CYP102A1 parent sequenceis assigned “1”, the CYP102A2 parent sequence is assigned “2”, and theCYP102A3 is parent sequence assigned “3”. In some instances, each parentsequence is divided into eight sequence blocks containing the followingamino acids (aa): block 1: aa 1-64; block 2: aa 65-122; block 3: aa123-166; block 4: aa 167-216; block 5: aa 217-268; block 6: aa 269-328;block 7: aa 329-404; and block 8: aa 405-end. Thus, in this example,there are eight blocks of amino acids and three fragments are possibleat each block. For instance, “12312312” refers to a chimeric P450protein of the invention containing block 1 (aa 1-64) from CYP102A1,block 2 (aa 65-122) from CYP102A2, block 3 (aa 123-166) from CYP102A3,block 4 (aa 167-216) from CYP102A1, block 5 (aa 217-268) from CYP102A2,and so on. Non-limiting examples of chimeric P450 proteins include thoseset forth in Table E (C2G9, X7, X7-12, C2E6, X7-9, C2B12, TSP234). Insome embodiments, the chimeric heme enzymes of the invention cancomprise at least one or more of the mutations described herein.

Chimeric Heme domain SEQ ID P450s block sequence NO C2G9 22223132 43 X722312333 44 X7-12 12112333 45 C2E6 11113311 46 X7-9 32312333 47 C2B1232313233 48 TSP234 22313333 49

An enzyme's total turnover number (or TTN) refers to the maximum numberof molecules of a substrate that the enzyme can convert before becominginactivated. In general, the TTN for the heme enzymes of the inventionrange from about 1 to about 100,000 or higher. For example, the TTN canbe from about 1 to about 1,000, or from about 1,000 to about 10,000, orfrom about 10,000 to about 100,000, or from about 50,000 to about100,000, or at least about 100,000. In particular embodiments, the TTNcan be from about 100 to about 10,000, or from about 10,000 to about50,000, or from about 5,000 to about 10,000, or from about 1,000 toabout 5,000, or from about 100 to about 1,000, or from about 250 toabout 1,000, or from about 100 to about 500, or at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000,20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000,65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, ormore. In certain embodiments, the variant or chimeric heme enzymes ofthe present invention have higher TTNs compared to the wild-typesequences. In some instances, the variant or chimeric heme enzymes haveTTNs greater than about 100 (e.g., at least about 100, 150, 200, 250,300, 325, 350, 400, 450, 500, or more) in carrying out in vitroaziridination reactions. In other instances, the variant or chimericheme enzymes have TTNs greater than about 1000 (e.g., at least about1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) incarrying out in vivo whole cell aziridination reactions.

When whole cells expressing a heme enzyme are used to carry out anaziridination reaction, the turnover can be expressed as the amount ofsubstrate that is converted to product by a given amount of cellularmaterial. In general, in vivo aziridination reactions exhibit turnoversfrom at least about 0.01 to at least about 10 mmol·g_(cdw) ⁻¹, whereing_(cdw) is the mass of cell dry weight in grams. For example, theturnover can be from about 0.1 to about 10 mmol·g_(cdw) ⁻¹, or fromabout 1 to about 10 mmol·g_(cdw) ⁻¹, or from about 5 to about 10mmol·g_(cdw) ⁻¹, or from about 0.01 to about 1 mmol·g_(cdw) ⁻¹, or fromabout 0.01 to about 0.1 mmol·g_(cdw) ⁻¹, or from about 0.1 to about 1mmol·g_(cdw) ⁻¹, or greater than 1 mmol·g_(cdw) ⁻¹. The turnover can beabout 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055,0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2,0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85,0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol·g_(cdw) ⁻¹.

When whole cells expressing a heme enzyme are used to carry out aaziridination reaction, the activity can further be expressed as aspecific productivity, e.g., concentration of product formed by a givenconcentration of cellular material per unit time, e.g., in g/L ofproduct per g/L of cellular material per hour (g g_(cdw) ⁻¹ h⁻¹). Ingeneral, in vivo aziridination reactions exhibit specific productivitiesfrom at least about 0.01 to at least about 0.5 g·g_(cdw) ⁻¹ h⁻¹, whereing_(cdw) is the mass of cell dry weight in grams. For example, thespecific productivity can be from about 0.01 to about 0.1 g g_(cdw) ⁻¹h⁻¹, or from about 0.1 to about 0.5 g g_(cdw) ⁻¹ h⁻¹, or greater than0.5 g g_(cdw) ⁻¹ h⁻¹. The specific productivity can be about 0.01,0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065,0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35,0.4, 0.45, or about 0.5 g g_(cdw) ⁻¹ h⁻¹.

In certain embodiments, mutations can be introduced into the target geneusing standard cloning techniques (e.g., site-directed mutagenesis) orby gene synthesis to produce the heme enzymes (e.g., cytochrome P450variants) of the present invention. The mutated gene can be expressed ina host cell (e.g., bacterial cell) using an expression vector under thecontrol of an inducible promoter or by means of chromosomal integrationunder the control of a constitutive promoter. Aziridination activity canbe screened in vivo or in vitro by following product formation by GC orHPLC as described herein.

The expression vector comprising a nucleic acid sequence that encodes aheme enzyme of the invention can be a viral vector, a plasmid, a phage,a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophageP1-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or anartificial chromosome (e.g., bacterial artificial chromosome (BAC), ayeast artificial chromosome (YAC), a mammalian artificial chromosome(MAC), and human artificial chromosome (HAC)). Expression vectors caninclude chromosomal, non-chromosomal, and synthetic DNA sequences.Equivalent expression vectors to those described herein are known in theart and will be apparent to the ordinarily skilled artisan.

The expression vector can include a nucleic acid sequence encoding aheme enzyme that is operably linked to a promoter, wherein the promotercomprises a viral, bacterial, archaeal, fungal, insect, or mammalianpromoter. In certain embodiments, the promoter is a constitutivepromoter. In some embodiments, the promoter is an inducible promoter. Inother embodiments, the promoter is a tissue-specific promoter or anenvironmentally regulated or a developmentally regulated promoter.

It is understood that affinity tags may be added to the N- and/orC-terminus of a heme enzyme expressed using an expression vector tofacilitate protein purification. Non-limiting examples of affinity tagsinclude metal binding tags such as His6-tags and other tags such asglutathione S-transferase (GST).

Non-limiting expression vectors for use in bacterial host cells includepCWori, pET vectors such as pET22 or pET22b(+) (EMD Millipore), pBR322(ATCC37017), pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene),pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1vectors, pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1(Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich,Mass.). Non-limiting examples of expression vectors for use ineukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR,pMT vectors (Life Technologies), pKLAC1 vectors, pKLAC2 vectors (NewEngland Biolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors,pAdeno-X™ adenoviral vectors (Clontech), and pBABE retroviral vectors.Any other vector may be used as long as it is replicable and viable inthe host cell.

The host cell can be a bacterial cell, an archaeal cell, a fungal cell,a yeast cell, an insect cell, or a mammalian cell.

Suitable bacterial host cells include, but are not limited to, BL21 E.coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 ExpressCompetent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescenscells, and cyanobacterial cells such as Chlamydomonas reinhardtii cellsand Synechococcus elongates cells. Non-limiting examples of archaealhost cells include Pyrococcus furiosus, Metallosphera sedula,Thermococcus litoralis, Methanobacterium thermoautotrophicum,Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus,Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungalhost cells include, but are not limited to, yeast cells from the generaSaccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces(e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cellsfrom the genera Aspergillus, Trichoderma, and Myceliophthora. Suitableinsect host cells include, but are not limited to, Sf9 cells fromSpodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Fivecells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cellsand Schneider 3 (S3) cells from Drosophila melanogaster. Non-limitingexamples of mammalian host cells include HEK293 cells, HeLa cells, CHOcells, COS cells, Jurkat cells, NS0 hybridoma cells, baby hamster kidney(BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any otherimmortalized cell line derived from a mammalian cell.

In certain embodiments, the present invention provides heme enzymes suchas the P450 variants described herein that are active aziridinationcatalysts inside living cells. As a non-limiting example, bacterialcells (e.g., E. coli) can be used as whole cell catalysts for the invivo aziridination reactions of the present invention. In someembodiments, whole cell catalysts containing a P450 enzymes variantdescribed herein significantly enhance the total turnover number (TTN)compared to in vitro reactions using isolated P450 enzymes.

B. Compounds

The methods of the invention can be used to provide a number ofaziridination products. The aziridination products described herein canbe useful starting materials or intermediates for the synthesis ofcompounds.

The olefinic substrates useful in the present invention are representedby a structure of Formula I:

For compounds of Formula I, R^(1a), R^(1b), and R² are independentlyselected from the group consisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl,aryl, heteroaryl, C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl,—Y¹-heteroaryl, —Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl; Y¹ isC₁₋₈alkylene; each R^(1a), R^(1b), and R² is optionally substituted withfrom 1 to 5 substituents independently selected from the groupconsisting of C₁₋₃alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido,oxo, thioxo, cyano, and halogen. In some embodiments, each aryl containsbetween 6-14 carbon atoms, each heteroaryl group has from 5 to 8 ringatoms and from 1-3 heteroatoms selected from N, O and S, and eachheterocyclyl group has from 1-3 heteroatoms selected from N, O and S.

In certain instances, R^(1a), R^(1b), and R² are independently selectedfrom the group consisting of H, C₁₋₁₈alkyl, aryl, heteroaryl,C₁₋₁₂cycloalkyl, and C₃₋₁₀heterocyclyl, each R^(1a), R^(1b), and R² isoptionally substituted with from 1 to 5 substituents independentlyselected from the group consisting of C₁₋₃alkyl, alkoxy, and halogen.

In some embodiments, R^(1a) is a substituted phenyl group or anaphthalenyl, wherein the phenyl group is substituted with 1 to 2 amethyl, chloro, or C₁alkyoxy groups.

In some embodiments, R^(1b) is H or methyl.

In some embodiments, R² is H or methyl. In some embodiments, R² is H.

The nitrene precursors useful in the present invention have a structureaccording to the Formula IIa or IIb:

wherein:

-   -   R³ is selected from the group consisting of C₁₋₁₈ alkyl,        C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,        C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b),        —PO₃R^(b)R^(c), and —CONR^(b)R^(c); X¹ is independently selected        from the group consisting of H and sodium, and X² is        independently selected from the group consisting of halogen,        —SO₂R^(a), —CO₂R^(b), —PO₃R^(b)R^(c), optionally X¹ and X² can        be taken together to form iodinane; R^(a) is independently        selected from the group consisting of C₁₋₈alkyl, hydroxy,        C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl, heteroaryl, and        C₃₋₈heterocyclyl; R^(b) and R^(c) are independently selected        from the group consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl,        heteroaryl, and C₃₋₈heterocyclyl; wherein within each R³, R^(a),        R^(b), and R^(c) can be optionally substituted with from 1-5        R^(d) substituents; and each R^(d) is independently selected        from the group consisting of C₁₋₃alkyl, halogen, and hydroxy. In        some embodiments, each aryl contains between 6-14 carbon atoms,        each heteroaryl group has from 5 to 10 ring atoms and from 1-3        heteroatoms selected from N, O and S, and each heterocyclyl        group has from 1-3 heteroatoms selected from N, O and S.

In certain instances, R³ is selected from the group consisting of aryl,—SO₂R^(a), —COR^(a), —CO₂R^(b), and —PO₃R^(b)R^(c); X¹ is independentlyselected from the group consisting of H and sodium, and X² isindependently selected from the group consisting of halogen, —SO₂R^(a),optionally X¹ and X² can be taken together to form iodinane; R^(a) isindependently selected from the group consisting of C₁₋₈alkyl,C₁₋₈alkoxy, and aryl; R^(b) and R^(c) are independently selected fromthe group consisting of C₁₋₈alkyl, and aryl; wherein within each R³,R^(a), R^(b), and R^(c) can be optionally substituted with from 1-5R^(d) substituents; and each R^(d) is independently selected from thegroup consisting of C₁₋₃alkyl, and halogen. In some embodiments, eacharyl contains between 6-14 carbon atoms, each heteroaryl group has from5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S,and each heterocyclyl group has from 1-3 heteroatoms selected from N, Oand S.

In some embodiments, the nitrene precursor has a structure selected fromthe group consisting of:

In some instances, the nitrene precursor is

In some embodiments, the aziridination product is a compound accordingto Formula III:

-   -   wherein R^(1a), R^(1b), and R² are independently selected from        the group consisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl, aryl,        heteroaryl, C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl,        —Y¹-heteroaryl, —Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl;        Y¹ is C₁₋₈alkylene; each R^(1a), R^(1b), and R² is optionally        substituted with from 1 to 5 substituents independently selected        from the group consisting of C₁₋₃alkyl, alkoxy hydroxyl, amino,        thiol, carboxy, amido, oxo, thioxo, cyano, and halogen; R³ is        selected from the group consisting of C₁₋₁₈ alkyl,        C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,        C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b),        —PO₃R^(b)R^(c), and —CONR^(b)R^(c); R^(a) is independently        selected from the group consisting of C₁₋₈alkyl, hydroxy,        C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl, heteroaryl, and        C₃₋₈heterocyclyl; R^(b) and R^(c) are independently selected        from the group consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl,        heteroaryl, and C₃₋₈heterocyclyl, wherein within each R³, R^(a),        R^(b), and R^(c) can be optionally substituted with from 1-5        R^(d) substituents; and each R^(d) is independently selected        from the group consisting of C₁₋₃alkyl, halogen, and hydroxy. In        some embodiments, each aryl contains between 6-14 carbon atoms,        each heteroaryl group has from 5 to 10 ring atoms and from 1-3        heteroatoms selected from N, O and S, and each heterocyclyl        group has from 1-3 heteroatoms selected from N, O and S.

In some instances, R^(1a) and R^(1b) are independently selected from thegroup consisting of H, C₁₋₈alkyl, aryl, heteroaryl, C₁₋₁₂cycloalkyl, andC₃₋₁₀heterocyclyl; R² is selected from the group consisting of H andC₁₋₈ alkyl; each R^(1a), R^(1b), and R² is optionally substituted withfrom 1 to 3 substituents independently selected from the groupconsisting of C₁₋₃alkyl, alkoxy, and halogen; and R³ is selected fromthe group consisting of —SO₂R^(a), —COR^(a), —CO₂R^(b), and—PO₃R^(b)R^(c); R^(a) is independently selected from the groupconsisting of C₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl;R^(b) and R^(c) are independently selected from the group consisting ofC₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl, wherein withineach R³, R^(a), R^(b), and R^(c) can be optionally substituted with from1-2 R^(d) substituents; and each R^(d) is independently selected fromthe group consisting of C₁₋₃alkyl, halogen, and hydroxy.

In some embodiments, the aziridination product is a compound accordingto Formula IIIa:

-   -   wherein R^(1a), R^(1b), and R² are independently selected from        the group consisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl, aryl,        heteroaryl, C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl,        —Y¹-heteroaryl, —Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl;        Y¹ is C₁₋₈alkylene; each R^(1a), R^(1b), and R² is optionally        substituted with from 1 to 5 substituents independently selected        from the group consisting of C₁₋₃alkyl, alkoxy hydroxyl, amino,        thiol, carboxy, amido, oxo, thioxo, cyano, and halogen; R³ is        selected from the group consisting of C₁₋₁₈ alkyl,        C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,        C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b),        —PO₃R^(b)R^(c), and —CONR^(b)R^(c); R^(a) is independently        selected from the group consisting of C₁₋₈alkyl, hydroxy,        C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl, heteroaryl, and        C₃₋₈heterocyclyl; R^(b) and R^(c) are independently selected        from the group consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl,        heteroaryl, and C₃₋₈heterocyclyl, wherein within each R³, R^(a),        R^(b), and R^(c) can be optionally substituted with from 1-5        R^(d) substituents; and each R^(d) is independently selected        from the group consisting of C₁₋₃alkyl, halogen, and hydroxy. In        some embodiments, each aryl contains between 6-14 carbon atoms,        each heteroaryl group has from 5 to 10 ring atoms and from 1-3        heteroatoms selected from N, O and S, and each heterocyclyl        group has from 1-3 heteroatoms selected from N, O and S.

In some instances, R^(1a) and R^(1b) are independently selected from thegroup consisting of H, C₁₋₈alkyl, aryl, heteroaryl, C₁₋₁₂cycloalkyl, andC₃₋₁₀heterocyclyl; R² is selected from the group consisting of H andC₁₋₈ alkyl; each R^(1a), R^(1b), and R² is optionally substituted withfrom 1 to 3 substituents independently selected from the groupconsisting of C₁₋₃alkyl, alkoxy, and halogen; and R³ is selected fromthe group consisting of —SO₂R^(a), —COR^(a), —CO₂R^(b), and—PO₃R^(b)R^(c); R^(a) is independently selected from the groupconsisting of C₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl;R^(b) and R^(c) are independently selected from the group consisting ofC₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl, wherein withineach R³, R^(a), R^(b), and R^(c) can be optionally substituted with from1-2 R^(d) substituents; and each R^(d) is independently selected fromthe group consisting of C₁₋₃alkyl, halogen, and hydroxy.

In some embodiments, compounds of Formula IIIa are further reactionproducts of an aziridine ring that has been opened after attack from anucleophile, such as a hydroxyl group. Compounds of formula IIIa can beproduced when the aziridination reactions described herein are performedunder aqueous reaction conditions.

In some embodiments, the aziridination product has a structure selectedfrom the group consisting of:

One of skill in the art will appreciate that stereochemicalconfiguration of certain of the products herein will be determined inpart by the orientation of the product of the enzymatic step. Certain ofthe products herein will be “cis” compounds or “Z” compounds. Otherproducts will be “trans” compounds or “E” compounds. One product wherecis or trans orientations are possible is the formation of an aziridinering. The cis configuration of an aziridine ring is when the highestpriority substituents are on the same side of the ring (e.g., FormulaIII when R^(1a) and R² are the highest priority substituents and on thesame side of the aziridine ring), while the trans configuration of anaziridine ring is when the highest priority substituents are on theopposite side of the ring.

In certain instances, two cis isomers and two trans isomers can arisefrom the reaction of an olefin substrate and a nitrene precursor. Thetwo cis isomers are enantiomers with respect to one another, in that thestructures are non-superimposable mirror images of each other.Similarly, the two trans isomers are enantiomers. One of skill in theart will appreciate that the absolute stereochemistry of a product—thatis, whether a given chiral center exhibits the right-handed “R”configuration or the left-handed “S” configuration—will depend onfactors including the structures of the particular substrate and nitreneprecursor used in the reaction, as well as the identity of the enzyme.The relative stereochemistry—that is, whether a product exhibits a cisor trans configuration—as well as for the distribution of productmixtures will also depend on such factors.

In certain instances, the product mixtures have cis:trans ratios rangingfrom about 1:99 to about 99:1. The cis:trans ratio can be, for example,from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or fromabout 1:50 to about 1:25, or from about 99:1 to about 75:1, or fromabout 75:1 to about 50:1, or from about 50:1 to about 25:1. Thecis:trans ratio can be from about 1:80 to about 1:20, or from about 1:60to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 toabout 40:1. The cis:trans ratio can be about 1:5, 1:10, 1:15, 1:20,1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80,1:85, 1:90, or about 1:95. The cis:trans ratio can be about 5:1, 10:1,15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1,75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of product mixture can be assessed in terms of theenantiomeric excess, or “% ee,” of the mixture. The enantiomeric excessrefers to the difference in the mole fractions of two enantiomers in amixture. Taking the reaction scheme in FIG. 8 as a non-limiting example,for instance, the enantiomeric excess of the “S” enantiomer can becalculated using the formula: %ee_(S)=[(χ_(S)−χ_(R))/(χ_(S)+χ_(R))]×100%, wherein χ is the molefraction for a given enantiomer. The enantiomeric excess of the “R”enantiomer (% ee_(R)) can be calculated in the same manner. In lessotherwise specified, % ee is reported as % ee_(S).

In general, product mixtures exhibit % ee values ranging from about 1%to about 99%, or from about −1% to about −99%. The closer a given % eevalue is to 99% (or −99%), the purer the reaction mixture is. The % eecan be, for example, from about −90% to about 90%, or from about −80% toabout 80%, or from about −70% to about 70%, or from about −60% to about60%, or from about −40% to about 40%, or from about −20% to about 20%.The % ee can be from about 1% to about 99%, or from about 20% to about80%, or from about 40% to about 60%, or from about 1% to about 25%, orfrom about 25% to about 50%, or from about 50% to about 75%. The % eecan be from about −1% to about −99%, or from about −20% to about −80%,or from about −40% to about −60%, or from about −1% to about −25%, orfrom about −25% to about −50%, or from about −50% to about −75%. The %ee can be about −99%, −95%, −90%, −85%, −80%, −75%, −70%, −65%, −60%,−55%, −50%, −45%, −40%, −35%, −30%, −25%, −20%, −15%, −10%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or about 95%. Any of these values can be % ee_(S) values or %ee_(R) values.

Accordingly, some embodiments of the invention provide methods forproducing a plurality of aziridination products having a % ee_(S) offrom about −90% to about 90%. In some embodiments, the % ee_(S) is atleast 90%. In some embodiments, the % ee_(S) is at least −99%. In someembodiments, the % ee_(R) is from about −90% to about 90%. In someembodiments, the % ee_(R) is at least 90%. In some embodiments, the %ee_(R) is at least −99%.

The methods of the disclosure can also be assessed in terms of thediastereoselectivity and/or enantioselectivity of the aziridinationreaction—that is, the extent to which the reaction produces a particularisomer, whether a diastereomer or enantiomer. A perfectly selectivereaction produces a single isomer, such that the isomer constitutes 100%of the product. As another non-limiting example, a reaction producing aparticular enantiomer constituting 90% of the total product can be saidto be 90% enantioselective. A reaction producing a particulardiastereomer constituting 30% of the total product, meanwhile, can besaid to be 30% diastereoselective. The diastereoselectivity and/orenantioselectivity of an aziridination reaction is dependent on a numberof factors including the olefinic substrate, nitrene precursor, and hemeenzyme used.

In general, the methods of the invention include reactions that are fromabout 1% to about 99% diastereoselective. The reactions are from about1% to about 99% enantioselective. The reaction can be, for example, fromabout 10% to about 90% diastereoselective, or from about 20%>to about80%>diastereoselective, or from about 40%>to about 60%)diastereoselective, or from about 1% to about 25% diastereoselective, orfrom about 25% o to about 50% diastereoselective, or from about 50% toabout 75% diastereoselective. The reaction can be about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orabout 95% diastereoselective. The reaction can be from about 10% toabout 90% enantioselective, from about 20% to about 80%enantioselective, or from about 40% to about 60% enantioselective, orfrom about 1% to about 25% enantioselective, or from about 25% to about50% enantioselective, or from about 50% to about 75% enantioselective.The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective.Accordingly some embodiments of the disclosure provide methods whereinthe reaction is at least 30% to at least 90% diastereoselective. In someembodiments, the reaction is at least 30% to at least 90%enantioselective.

C. Reaction Conditions

The methods of the invention include forming reaction mixtures thatcontain the heme enzymes described herein. The heme enzymes can be, forexample, purified prior to addition to a reaction mixture or secreted bya cell present in the reaction mixture. The reaction mixture can containa cell lysate including the enzyme, as well as other proteins and othercellular materials. Alternatively, a heme enzyme can catalyze thereaction within a cell expressing the heme enzyme. Any suitable amountof heme enzyme can be used in the methods of the invention. In general,aziridination reaction mixtures contain from about 0.01 mol % to about10 mol % heme enzyme with respect to the nitrene precursor and/orolefinic substrate. The reaction mixtures can contain, for example, fromabout 0.01 mol % to about 0.1 mol % heme enzyme, or from about 0.1 mol %to about 1 mol % heme enzyme, or from about 1 mol % to about 10 mol %heme enzyme. The reaction mixtures can contain from about 0.05 mol % toabout 5 mol % heme enzyme, or from about 0.05 mol % to about 0.5 mol %heme enzyme. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, or about 1 mol % heme enzyme.

The concentration of olefinic substrate and nitrene precursor aretypically in the range of from about 100 μM to about 1 M. Theconcentration can be, for example, from about 100 μM to about 1 mM, orabout from 1 mM to about 100 mM, or from about 100 mM to about 500 mM,or from about 500 mM to 1 M. The concentration can be from about 500 μMto about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about 50mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30mM. The concentration of olefinic substrate or nitrene precursor can be,for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM.The concentration of olefinic substrate or nitrene precursor can beabout 1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500mM.

Reaction mixtures can contain additional reagents. As non-limitingexamples, the reaction mixtures can contain buffers (e.g.,2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS),2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate,sodium phosphate, phosphate-buffered saline, sodium citrate, sodiumacetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, isopropanol, glycerol,tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g.,NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., ureaand guandinium hydrochloride), detergents (e.g., sodium dodecylsulfateand Triton-X 100), chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducingagents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT),3-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)).Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars,and reducing agents can be used at any suitable concentration, which canbe readily determined by one of skill in the art. In general, buffers,cosolvents, salts, denaturants, detergents, chelators, sugars, andreducing agents, if present, are included in reaction mixtures atconcentrations ranging from about 1 μM to about 1 M. For example, abuffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, asugar, or a reducing agent can be included in a reaction mixture at aconcentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, orabout 250 mM, or about 500 mM, or about 1 M. In some embodiments, areducing agent is used in a sub-stoichiometric amount with respect tothe olefin substrate and the nitrene precursor. Cosolvents, inparticular, can be included in the reaction mixtures in amounts rangingfrom about 1% v/v to about 75% v/v, or higher. A cosolvent can beincluded in the reaction mixture, for example, in an amount of about 5,10, 20, 30, 40, or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze theformation of an aziridination product. The reactions can be conducted atany suitable temperature. In general, the reactions are conducted at atemperature of from about 4° C. to about 40° C. The reactions can beconducted, for example, at about 25° C. or about 37° C. The reactionscan be conducted at any suitable pH. In general, the reactions areconducted at a pH of from about 6 to about 10. The reactions can beconducted, for example, at a pH of from about 6.5 to about 9. Thereactions can be conducted for any suitable length of time. In general,the reaction mixtures are incubated under suitable conditions foranywhere between about 1 minute and several hours. The reactions can beconducted, for example, for about 1 minute, or about 5 minutes, or about10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, orabout 4 hours, or about 8 hours, or about 12 hours, or about 24 hours,or about 48 hours, or about 72 hours. Reactions can be conducted underaerobic conditions or anaerobic conditions. Reactions can be conductedunder an inert atmosphere, such as a nitrogen atmosphere or argonatmosphere. In some embodiments, a solvent is added to the reactionmixture. In some embodiments, the solvent forms a second phase, and theaziridination reaction occurs in the aqueous phase. In some embodiments,the heme enzymes is located in the aqueous layer whereas the substratesand/or products occur in an organic layer. Other reaction conditions maybe employed in the methods of the invention, depending on the identityof a particular heme enzyme, olefinic substrate, or nitrene precursor.

Reactions can be conducted in vivo with intact cells expressing a hemeenzyme of the invention. The in vivo reactions can be conducted with anyof the host cells used for expression of the heme enzymes, as describedherein. A suspension of cells can be formed in a suitable mediumsupplemented with nutrients (such as mineral micronutrients, glucose andother fuel sources, and the like). Aziridination yields from reactionsin vivo can be controlled, in part, by controlling the cell density inthe reaction mixtures. Cellular suspensions exhibiting optical densitiesranging from about 0.1 to about 50 at 600 nm can be used foraziridination reactions. Other densities can be useful, depending on thecell type, specific heme enzymes, or other factors.

IV. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Aziridination Activity of Cytochrome P450 Variants and OtherHeme Proteins

This example illustrates the aziridination activity of known cytochromeP450 variants and other heme containing enzymes.

Previous studies have shown that cytochrome P450 and mutants thereof cancatalyze a wide variety of chemical reactions includingcyclopropanation, sulfinde imidation, and C—H amination. In order toassess the potential of a cytochrome P450 or a mutant thereof tocatalyze an aziridination reaction, engineered variants of cytochromeP450_(BM3) and P411_(BM3)-CIS-T438S, previously found to be effectivefor intramolecular C—H amination and sulfide imidation, were tested foraziridination activity. Cytochrome P450_(BM3) is a naturally occurringenzyme found in the soil bacterium bacillus megaterium, andP411_(BM3)-CIS-T438S is a 14 mutation variant of P450_(BM3) (see Table 2for mutations from wild-type P450_(BM3)). P411_(BM3)-CIS-T438S is calleda “P411” due to the change in the characteristic CO-bound Soret peakfrom 450 to 411 nm effected by mutation of the cysteine residue thatcoordinates the heme iron to serine (C400S). This axial cysteine iscompletely conserved in cytochrome P450s and is required for the nativemonooxygenase activity. Thus, the P411 enzyme is no longer a “cytochromeP450”, nor does it exhibit its native hydroxylation activity. However,the C400S mutation increases the non-natural carbene transfer activitiesof P450_(BM3) and other P450s. Two crystal structures of P411 variantsof P450_(BM3) show that S400 coordinates the iron and causes nosignificant structural perturbation in the substrate binding pocket.

The aziridination activity of P411_(BM3)-CIS-T438S was tested usingstyrene derivatives as the olefin substrate and tosyl azide (TsN₃) asthe nitrene precursor (Table 1). Tosyl azide was completely consumed inthis reaction, the major product of which was the azide reductionproduct p-toluenesulfonamide (>300 total turnovers (TTN), not shown inTable 1). Amidoalcohol 2 appeared as a minor product. Controlexperiments showed that the desired aziridine product rapidly decomposesunder aqueous reaction conditions to the corresponding amidoalcohol 2(FIGS. 2A-B, 3A-D, and 4A-D). Degradation of this aziridine product hasalso been observed in studies with small-molecule catalysts (Ando, T.;et al., Tetrahedron 54, 13485-13494 (1998) and Kiyokawa, K. et al., Org.Lett., 15, 4858-4861 (2013)). It was thus inferred that production of 2is directly related to the nitrene transfer activity of the enzymetoward olefin 1.

TABLE 1 Total turnovers (TTN) to product for aziridination catalyzed bypurified holoenzymes P411_(BM3)-CIS-T438S (P) andP411_(BM3)-CIS-T438S-I263F (P-I263F) with selected styrenyl olefins 1,3, and 5 and tosyl azide.^(a)

Enzyme TTN 2 ^(b) TTN 4 TTN 6 P411_(BM3)-CIS-T438S (P) 15 8 5 P-I263F150 160 190 ^(a)Reactions were performed in 0.1 M KPi buffer pH = 8.0using 0.2 mol % enzyme and NADPH as reductant, with 2.5 mM tosyl azideand 7.5 mM olefin. Detailed reaction conditions can be found in thesupporting information.^(b) TTN=Total turnover number. TTNs were determined by HPLC analysis.

This low level of nitrene transfer activity to 4-methoxystyrene olefinof the P411_(BM3)-CIS-T438S enzyme prompted investigation of othervariants. A small set of cytochrome P450_(BM3) variants and hemeproteins prepared for other studies were chosen in order to assess howchanges in the protein sequence affect nitrene transfer to olefinsubstrates. Table 2 shows the variants of the cytochrome P450_(BM3)mutants tested, and Tables 3 and 4 illustrate the results of thesetests.

TABLE 2 Mutations present in P450 BM3 variants tested. Enzyme Mutationsrelative to wild-type P450_(BM3) P450_(BM3) none P450_(BM3)-CIS V78A,F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V,I366V, E442K P450_(BM3)-CIS T438S P450_(BM3)-CIS T438S P450_(BM3)-CIST438S C400H P450_(BM3)-CIS T438S, C400H P450_(BM3)-CIS T438S C400DP450_(BM3)-CIS T438S, C400D P450_(BM3)-CIS T438S C400M P450_(BM3)-CIST438S, C400M P411_(BM3)-CIS P450_(BM3)-CIS C400S P411_(BM3)-CIS T438SP450_(BM3)-CIS T438S, C400S P411_(BM3)-CIS A268T T438S P450_(BM3)-CISA268T, C400S, T438S P411_(BM3)-H2-5-F10 P450_(BM3)-CIS L75A, I263A,C400S, L437A P411_(BM3)-H2-A-10 P450_(BM3)-CIS L75A, L181A, C400SP411_(BM3)-H2-4-D4 P450_(BM3)-CIS L75A, M177A, L181A, C400S, L437AP411_(BM3) P450_(BM3)-C400S P450_(BM3)-T268A T268A P411_(BM3)-T268AP450_(BM3)-T268A, C400S P411_(BM3)-CIS T438S I263F P450_(BM3)-CIS T438S,I263F, C400S (P-I263F) P411_(BM3)-CIS T438S I263F P450_(BM3)-CIS T438S,I263F, C400S, V87F V87F P411_(BM3)-CIS T438S I263F P450_(BM3)-CIS T438S,I263F, C400S, A268T A268T

TABLE 3 Panel of P450_(BM3) purified enzymes tested for aziridinationreactivity with 4-methoxystyrene and tosyl azide.^(a)

Entry Enzyme TTN 2 1 P411_(BM3)-CIS T438S (P) 15 2 P450_(BM3)-CIS T438S<1 3 P450_(BM3)-CIS T438S C400H 3 4 P450_(BM3)-CIS T438S C400D 4 5P450_(BM3)-CIS T438S C400M 4 6 P411_(BM3)-CIS A268T T438S <1 7P411_(BM3)-H2-5-F10 8 8 P411_(BM3)-H2-A-10 4 9 P411_(BM3)-H2-4-D4 5 10P450_(BM3) <1 11 P411_(BM3) 3 12 P450_(BM3)-T268A 2 13 P411_(BM3)-T268A4 14 P411_(BM3)-CIS T438S I263F (P-I263F) 150 14 P411_(BM3)-CIS T438SI263F V87F 19 15 P411_(BM3)-CIS T438S I263F A268T <1 ^(a)“P411” denotesSer-mutated (C400S) variant of cytochrome P450_(BM3). Variant IDs andspecific amino acid substitutions in each can be found in Table 2.TTN—total turnover number.

TABLE 4 Heme and other heme-containing proteins tested for activity inthe above reaction (Table 3) with 4-methoxystyrene. Myoglobin andcytochrome c were purchased as lyophilized powder from Sigma Aldrich.P450Rhf mutants were expressed and purified as described in the methodssection; P450CYP119 was expressed and purified as described in Heel, T.et al., ChemBioChem., 15, 2556(2014). Entry Catalyst TTN 2 1 Hemin <1 2Hemin + BSA <1 3 Myoglobin (horse heart) <1 4 Oytochrome c (bovineheart) <1 5 CYP119 C317S 7 6 CYP119 T213A C317H <1 7 P450_(Rhf) <1

P450_(BM3) sequences lacking the C400S and/or T268A mutations were notactive, nor did the Fe(II)-protoporphyrin IX (PPIX) cofactor catalyzeaziridination under these conditions. Mutants differing fromP411_(BM3)-CIS-T438S by 2-5 alanine mutations in the active site showedsome aziridination activity (4-8 TTN), but none was more productive thanP411_(BM3)-CIST438S. A set of enzymes containing different axialmutations were tested, including the S400H, S400D, and S400M mutants ofP411_(BM3)-CIS-T438S. These enzymes were also only weakly active, giving2 at levels lower than P411_(BM3)-CIS-T438S (3-4 TTN). Myoglobin (horseheart), cytochrome c (bovine heart), and cytochrome P450_(Rhf) (fromRhodococcus sp. NCIMB 9784) were all inactive for this intermolecularaziridination (Table 4). An engineered variant of the thermostablecytochrome P450 from Sulfolobus acidocaldarius, CYP119, that containedan axial cysteine-to-serine mutation (C317S) did catalyze low levels ofaziridination (˜7 TTN). This demonstrates that mutations previouslydescribed to activate non-natural nitrene-transfer activity inP450_(BM3) can confer measurable activity on other P450s as well.

Of all the enzymes tested, a variant of P411BM3-CIS-T438S having asingle active-site substitution, I263F, was the most active toward4-methoxystyrene, providing 150 total turnovers in the formation ofamido-alcohol 2 from 4-methoxystyrene (Table 3). P-I263F was even moreproductive when the reactions were carried out using whole Escherichiacoli cells expressing this enzyme (FIG. 5), consistent with our previousobservations that enzyme-catalyzed metal-nitrenoid and metal-carbenoidtransfer activities improved when the reactions were performed withwhole cells. No aziridine product was observed when cells not expressingthe P411 catalyst were used.

Example 2 Optimizing Cytochrome P450 Aziridination Activity

This example illustrates bacterial cytochrome P450s that are engineeredto catalyze highly stereoselective nitrene transfers to olefinsubstrates to make aziridines.

The P-I263F enzyme identified in the initial studies of enzyme catalyzedaziridination provided enough aziridine product in whole-cell reactionsto allow for screening variants in 96-well plate format. Thus, furtherimprovement of aziridination productivity was sought by mutagenesis ofthis enzyme and screening for aziridination productivity.Site-saturation mutagenesis (SSM) libraries were created at severalactive site positions that were previously shown to influenceproductivity and enantioselectivity in other non-natural reactions (A78,L181, T438, A328). Screening of these single SSM libraries foraziridination of 4-methylstyrene (3) identified P-I263F-A328V, withslightly improved yield and substantially improved % ee (96% ee_(S);entry 4, Table 5). Another round of SSM performed on this variant atadditional active site positions (F87, T268, L437) resulted inP-I263F-A328V-L437V with improved aziridine yield and a further increasein enantioselectivity (99% ee_(S)). The P-I263F-L437V and P-I263F-A328Vmutants were both less selective than P-I263F-A328V-L437V, demonstratingthat both new mutations contribute to the very high enantioselectivity.Importantly, the yield of sulfonamide side product 7 diminished over thecourse of active site evolution, to the extent that aziridine 4 becamethe major product of the reaction catalyzed by P-I263F-A328V-L437V.

TABLE 5 Improvement in yield and % ee for aziridine product 4 withactive-site evolution of P411_(BM3)CIS-T438S (P).^(a)

Entry Enzyme % yield 4 % yield 7 % ee 4 1 No enzyme 0 95 nd 2P411_(BM3)-CIS-T438S 1.1 95 25 3 P-I263F 40 54 55 4 P-I263F-A328V 43 5096 5 P-I263F-L437V 37 52 95 6 P-I263F-A328V-L437V 55 43 99 ^(a)Reactionswere carried out using whole E. coli cells resuspended in M9-N reactionbuffer under anaerobic conditions, with 2.5 mM tosyl azide and 7.5 mM4-methylstyrene. Yield is based on tosyl azide. See methods for detailedreaction set up and quantification procedures. ^(b)% ee determined bySFC analysis and calculated as (S − R) / (S + R). ^(c)‘No enzyme’reactions were carried out using whole cells with no P411 enzymeexpressed, as described in the SI methods

Because the azide is fully consumed in these reactions, the improvedaziridine yield could result from either an increase in the rate ofaziridine formation or a decrease in the rate of competing azidereduction, or from a combination of both. To address this, initial ratesof reaction were measured with the PI263F, P-I263F-A328V, andP-I263F-A328V-L437V enzymes as purified holoenzymes (FIGS. 6 and 7A-C).Initial rates of aziridination for the purified enzymes reflected theyield improvements observed in whole cells: P-I263F and P-I263FA328Vhave similar turnover frequencies (15-16 min⁻¹), whileP-I263F-A328V-L437V, having both new mutations, was improved (TOF ˜24min⁻¹). The initial turnover frequency of sulfonamide formation in vitrowas similar for all the enzymes, and faster than aziridine formation(TOFs ˜26-29 min⁻¹.

Example 3 Productivity and Enantioselectivity of Select Cytochrome P450Enzymes

This example illustrates the aziridination productivity andenantioselectivity of P-I263F-A328V-L437V when reacted with differentsubstrates. This example also illustrates the aziridination productivityand enantioselectivity using enzyme variant P411_(BM3) H2-A-10 I263F.

Having obtained a variant capable of high productivity andenantioselectivity for the aziridination of 4-methylstyrene (3),whole-cell reactions with different substituted styrene substrates wereinvestigated (Table 6). No correlation between the electronics of thearyl substituent and the productivity of the enzyme were observed. Ingeneral, the evolved enzyme was more productive with styrenessubstituted at the 4-position, though the highest productivity wasobserved with styrene itself. The evolved enzyme provided 600 catalyticturnovers for the formation of aziridine 6, corresponding to a 70% yieldof 6 (entry 3 in Table 6). With higher styrene and tosyl azide loading,the enzyme catalyzed 1,000 turnovers for aziridination, while retaininghigh (S)-selectivity (99% ee) (FIG. 8). Both 3-methylstyrene and3-chlorostyrene were significantly less reactive than their4-substituted counterparts, giving 85 and 21 turnovers, respectively,compared to 450 and 290 turnovers (entries 2, 4, 5, 6 in Table 6). Theevolved enzyme is an exceptionally enantioselective aziridinationcatalyst with styrene entries 2-4 (Table 6), giving 99% ee in favor ofthe (S)-enantiomer with these three substrates. Both 4-methoxystyreneand α-methylstyrene (entries 1 and 8 in Table 6) gave exclusivelyracemic amido-alcohol product. Formation of the amido-alcohol productfrom these substrates may result from carbocation stabilization at thebenzylic position due to the resonance and hyperconjugativestabilization provided by the respective p-OMe and α-Me groups, leadingto decomposition of the aziridine product and subsequent carbocationquenching with water.

TABLE 6 Substrate aziridination with P-I263F-A328V-L437V showingproductivity in terms of TTN and selectivity in % ee for eachproduct.^(a) Entry Olefin Product TTN % yield % ee ^(b) 1

390 47 rac 2

450 55 99 3

600 70 99 4

290 36 99 5

21 2 95 6

85 10 95 7

130 15 81 8

83 10 rac 9

53 6 88 ^(a)Reactions were carried out with whole cells expressingP-I263F-A328V-L437V under anaerobic conditions, with 2.5 mM tosyl azideand 7.5 mM olefin. Reactions were allowed to proceed for 4 hours at roomtemperature. ^(b) % ee determined as (S − R) / (S + R). Absoluteconfigurations were assigned based on analogy to 6. rac = racemic.

Although previous work has highlighted the importance of modulating hemeelectronic properties to access non-natural reactivity (McIntosh, J. A.;et al., Angew. Chem., Int. Ed. 52, 9309-9312 (2013); Hyster, T. K.; etal., J. Am. Chem. Soc., 136, 15505-15508 (2014); Coelho, P. S.; et al.,Nat. Chem. Biol. 9, 485-487 (2013)), here it was observed that stronggains in aziridination activity are brought about by mutations on thedistal heme side, suggesting that their effect may be the result ofimproving substrate binding and orientation, a hallmark of enzymecatalysis that is notable for a new-to-nature reaction such asP450-catalyzed nitrene transfer.

P-I263F-A328V-L437V is an exceptionally (S)-selective aziridinationcatalyst with olefin entries 2-4 (Table 6), giving 99% ee in favor ofthe (S)-enantiomer with these three substrates. Also identified in thiswork is the P411_(BM3) H2-A-10 I263F enzyme variant which is an I263Fmutant of the P411_(BM3) H2-A-10 enzyme identified in a previous study.The P411_(BM3) H2-A-10 I263F enzyme is able to catalyze theaziridination reaction with enantioselectivity that favors theR-enantiomer (84% ee in favor of (R)-enantiomer, see reaction schemebelow).

Example 4 Synthesis of Substrates and Standards

The following example illustrates the synthesis of substrates andstandards.

N-(2-hydroxy-2-(4-methoxyphenyl)ethyl)-4-methylbenzenesulfonamide (2)

Synthesized as previously reported in Srinivas, B. et al., J. Mol.Catal. A: Chem., 261, 1-5 (2007).

¹H NMR (400 MHz, CDCl₃): δ 7.72 (d, 2H, J=8.1 Hz), 7.29 (d, 2H, J=8.3Hz), 7.19 (d, 2H, J=8.6 Hz), 6.84 (d, 2H, 8.6 Hz), 5.06 (dd, 1H, J=8.1,4.6 Hz), 4.73 (dd, 1H, J=8.7, 3.7 Hz), 3.78 (s, 3H), 3.20 (ddd, 1H,J=13.3, 8.1, 3.7 Hz), 3.01 (ddd, 1H, J=13.2, 8.6, 4.6 Hz), 2.42 (s, 3H)

¹³C NMR (101 MHz, CDCl₃): δ 159.66, 143.69, 136.86, 133.00, 129.90,127.26, 127.21, 114.16, 72.50, 55.44, 50.30, 21.66

HRMS (FAB+): calculated for C₁₆H₁₈NO₄S ([M+H]+): 320.0956. found:320.0950.

N-(p-Tolylsulfonyl)-2-(p-methylphenyl)aziridine (4)

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Huang, C. Y. et al., J. Am. Chem. Soc., 134, 9541-9544(2012); Kiyokawa, K. et al., Org. Lett. 15, 4858-4861 (2013); Evans. D.et al., J. Am. Chem. Soc., 116, 2742-2753 (1994)).

¹H NMR (300 MHz, CDCl₃): δ 7.86 (d, 2H, J=8.3 Hz), 7.32 (d, 2H, J=8.3Hz), 7.10 (s, 4H), 3.74 (dd, 1H, J=7.2, 4.5 Hz), 2.97 (d, 1H, J=7.2 Hz),2.43 (s, 3H), 2.38 (d, 1H, J=4.5 Hz), 2.31 (s, 3H).

N-(p-Tolylsulfonyl)-2-phenylaziridine (6)

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Huang, C. Y. et al., J. Am. Chem. Soc., 134, 9541-9544(2012); Kiyokawa, K. et al., Org. Lett. 15, 4858-4861 (2013); Evans. D.et al., J. Am. Chem. Soc., 116, 2742-2753 (1994)).

¹H NMR (300 MHz, CDCl₃): δ 7.87 (d, 2H, J=8.3 Hz), 7.19-7.36 (m, 7H),3.77 (dd, 1H, J=7.2, 4.5 Hz), 2.98 (d, 1H, J=7.2 Hz), 2.43 (s, 3H), 2.39(d, 1H, J=4.5 Hz)

N-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Huang, C. Y. et al., J. Am. Chem. Soc., 134, 9541-9544(2012); Kiyokawa, K. et al., Org. Lett. 15, 4858-4861 (2013); Evans. D.et al., J. Am. Chem. Soc., 116, 2742-2753 (1994)).

¹H NMR (500 MHz, CDCl₃): δ 7.87 (d, 2H, J=8.3 Hz), 7.34 (d, 2H, J=8.5Hz), 7.14 (d, J=8.7 Hz, 2H), 6.83 (d, J=8.7, 2H), 3.78 (s, 3H), 3.75(dd, 1H, J=7.2, 4.5 Hz), 2.97 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.39 (d,1H, J=4.5 Hz)

N-(p-Tolylsulfonyl)-2-(p-chlorophenyl)aziridine

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Huang, C. Y. et al., J. Am. Chem. Soc., 134, 9541-9544(2012); Kiyokawa, K. et al., Org. Lett. 15, 4858-4861 (2013); Evans. D.et al., J. Am. Chem. Soc., 116, 2742-2753 (1994)).

¹H NMR (300 MHz, CDCl₃): δ 7.86 (d, 2H, J=8.3 Hz), 7.34 (d, 2H, J=7.9Hz), 7.26 (d, 2H, J=8.5 Hz), 7.15 (d, 2H, J=8.5 Hz), 3.73 (dd, 1H,J=7.2, 4.4 Hz), 2.98 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.34 (d, 1H, J=4.4Hz)

N-(p-Tolylsulfonyl)-2-(m-chlorophenyl)aziridine

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Craig II, R. A.; et al. Chem. Eur. J., 20, 4806-4813(2014)).

¹H NMR (400 MHz, CDCl₃): δ 7.87 (d, 2H, J=8.3 Hz), 7.35 (d, 2H, J=7.7Hz), 7.19-7.26 (m, 3H), 7.12 (dt, 1H, J=6.8, 1.8 Hz), 3.73 (dd, 1H,J=7.2, 4.3 Hz), 2.97 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.35 (d, 1H, J=4.4Hz)

N-(p-Tolylsulfonyl)-2-(m-methylphenyl)aziridine

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Gao, G. Y. et al., Org. Lett., 7, 3191-3193 (2005)).

¹H NMR (400 MHz, CDCl₃): δ 7.87 (d, 2H, J=8.3 Hz), 7.33 (d, 2H, J=8.6Hz), 7.01-7.20 (m, 4H), 3.74 (dd, 1H, J=7.2, 4.5 Hz), 2.96 (d, 1H, J=7.2Hz), 2.43 (s, 3H), 2.38 (d, 1H, J=4.5 Hz), 2.30 (s, 3H)

N-(p-Tolylsulfonyl)-2-(2,4-dimethylphenyl)aziridine

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998).

¹H NMR (400 MHz, CDCl₃): δ 7.90 (d, 2H, J=8.4 Hz), 7.34 (d, 2H, J=8.5Hz), 6.91-7.00 (m, 3H), 3.84 (dd, 1H, J=7.2, 4.6 Hz), 2.97 (d, 1H, J=7.2Hz), 2.44 (s, 3H), 2.35 (s, 3H), 2.32 (d, 1H, J=4.6 Hz), 2.28 (s, 3H)

¹³C NMR (101 MHz, CDCl₃): δ 144.72, 137.95, 136.72, 135.15, 130.89,130.32, 129.84, 128.11, 126.82, 125.98, 39.61, 35.07, 21.75, 21.11,19.08

HRMS (FAB+): calculated for C₁₇H₂₀NO₂S ([M+H]+): 302.1215. found:302.1210.

N-(2-hydroxy-2-phenylpropyl)-4-methylbenzenesulfonamide

Synthesized as previously reported in Srinivas, B. et al., J. Mol.Catal. A: Chem., 261, 1-5 (2007).

¹H NMR (400 MHz, CDCl₃): δ 7.67 (d, 2H, J=8.3 Hz), 7.24-7.38 (m, 7H),4.59 (s, 1H), 3.22 (dd, 1H, J=12.8, 8.5 Hz), 3.12 (dd, 1H, J=12.8, 4.8Hz), 2.42 (s, 3H), 1.56 (s, 3H)

¹³C NMR (101 MHz, CDCl₃): δ 144.87, 143.73, 136.73, 129.93, 128.75,127.60, 127.19, 124.93, 73.81, 53.99, 27.62, 21.68

HRMS (FAB+): calculated for C₁₆H₂₀NO₃S ([M+H]+): 306.1164. found:306.1160.

N-(p-Tolylsulfonyl)-2-(naphthalene-2-yl)aziridine

Synthesized as previously reported in Ando, T et al., Tetrahedron, 54,13485-13494 (1998) with spectral data in agreement with literaturereported values (Huang, C. Y. et al., J. Am. Chem. Soc., 134, 9541-9544(2012); Kiyokawa, K. et al., Org. Lett. 15, 4858-4861 (2013); Evans. D.et al., J. Am. Chem. Soc., 116, 2742-2753 (1994)).

¹H NMR (400 MHz, CDCl₃): δ 7.90 (d, 2H, J=8.3 Hz), 7.75-7.81 (m, 3H),7.73 (s, 1H), 7.45-7.49 (m, 2H), 7.33 (d, 2H, J=8.3 Hz), 7.25-7.30 (m,1H), 3.93 (dd, 1H, J=7.2, 4.4 Hz), 3.07 (d, 1H, J=7.2 Hz), 2.50 (d, 1H,J=4.5 Hz), 2.42 (s, 3H)

Materials and Methods

The following paragraphs describe in more detail the materials andmethods used in Examples 1-3.

General.

Unless otherwise noted, all chemicals and reagents for reactions wereobtained from commercial suppliers (Sigma-Aldrich, VWR, Alfa Aesar) andused without further purification. Silica gel chromatographypurifications were carried out using AMD Silica Gel 60, 230-400 mesh. ¹Hspectra were recorded on a Varian Inova 300 MHz or Bruker Prodigy 400MHz instrument in CDCl₃, and are referenced to the residual solventpeak. Synthetic reactions were monitored using thin layer chromatography(Merck 60 gel plates) using an UV-lamp for visualization.

Chromatography.

Analytical high-performance liquid chromatography (HPLC) was carried outusing an Agilent 1200 series, and a Kromasil 100 C18 column (4.6×50 mm,5 μm). Semi-preparative HPLC was performed using an Agilent XDB-C18(9.4×250 mm, 5 μm). Analytical chiral HPLC was conducted using asupercritical fluid chromatography (SFC) system with isopropanol andliquid CO₂ as the mobile phase. Chiral OB-H and AS-H columns were usedto separate aziridine and amido-alcohol enantiomers (4.6×150 mm, 5 μm).Olefins were all commercially available; amido-alcohol and aziridinestandards were prepared as reported. % ee was calculated by dividing themajor peak area by the sum of the peak areas determined by SFCchromatography

Cloning and Site-Directed Mutagenesis.

pET22b(+) was used as a cloning and expression vector for all enzymesdescribed in this study. Site-directed mutagenesis was performed using amodified QuikChange™ mutagenesis protocol. The PCR products were gelpurified, digested with DpnI, repaired using Gibson Mix™, and directedtransformed into E. coli strain BL21(DE3).

Determination of P450 Concentration.

Concentration of P450/P411 enzymes for in whole cell experiments wasdetermined from ferrous carbon monoxide binding difference spectra usingpreviously reported extinction coefficients for cysteine-ligated(c=91,000 M⁻¹ cm⁻¹) and serine-ligated enzymes (c=103,000 M⁻¹ cm⁻¹).When purified enzymes were used, concentration of P450/P411 enzymes wasaccomplished by quantifying the amount of free hemin present in purifiedprotein using the pyridine/hemochrome assay.

Protein Expression and Purification.

Enzymes used in purified protein experiments were expressed in BL21(DE3)E. coli cultures transformed with plasmid encoding P450 or P411variants. Expression and purification were performed as described exceptthat the shake rate was lowered to 130 RPM during expression (Coelho, P.S., et al. Science, 339, 307 (2013)). Following expression, cells werepelleted and frozen at −20 OC. For purification, frozen cells wereresuspended in buffer A (20 mM tris, 20 mM imidazole, 100 mM NaCl, pH7.5, 4 mL/g of cell wet weight), loaded with 300 μg/ml hemin, anddisrupted by sonication (2×1 min, output control 5, 50% duty cycle;Sonicator 3000, Misonix, Inc.). To pellet insoluble material, lysateswere centrifuged (20,000×g for 0.5 h at 4° C.). Proteins were expressedin a construct containing a 6×-His tag and were consequently purifiedusing a nickel NTA column (1 mL HisTrap HP, GE Healthcare, Piscataway,N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). P450 orP411 enzymes were then eluted on a linear gradient from 0% buffer B (20mM tris, 300 mM imidazole, 100 mM NaCl, pH 7.5) to 100% buffer B over 10column volumes (P450/P411 enzymes elute at around 80 mM imidazole).Fractions containing P450 or P411 enzymes were pooled, concentrated, andsubjected to three exchanges of phosphate buffer (0.1 M KPi pH 8.0) toremove excess salt and imidazole. Concentrated proteins were aliquoted,flash-frozen on powdered dry ice, and stored at −20° C. until later use

Reaction Screening in 96-Well Plate Format.

Site-saturation mutagenesis libraries were generated by employing the“22c-trick” method (Kille, S., et al., ACS Synth. Biol., 2, 83-92(2013)). E. coli libraries were generated and cultured in 300 μL of LBwith 100 ug/ml ampicillin and stored as glycerol stocks at −80° C. in96-well plates. 50 μL of the pre-culture was transferred to a 1000 μL ofHyperbroth using a multichannel pipette. The cultures were incubated at37° C., 220 rpm, 80% humidity for 3 hours. The plates were cooled on icefor 15 minutes before expression was induced (0.5 mM IPTG, 1 mM5-aminolevulinic acid final concentration). Expression was conducted at20° C., 120 rpm, 20 h. The cells were pelleted (3000×g, 5 min) andre-suspended in 40 μL/well GOX solution (14,000 U/ml catalase (Sigma02071) and 1000 U/ml glucose oxidase (Sigma G7141)). The 96-well platewas transferred to an anaerobic chamber. To this mixture was added 300μL per well argon sparged reaction buffer (4:1 M9-N: 250 mM glucose inM9-N) was added followed by 4-methylstyrene (300 mM, 10 μL/well) andtosyl azide (100 mM, 10 μL/well). The plate was sealed with aluminumsealing tape, removed from the anaerobic chamber, and shaken at 40 rpm.After 16 hours, the seal was removed and 400 μL of acetonitrile wasadded to each well. The contents of each well were mixed by pipetting upand down using a multichannel pipette. Then the plate was centrifuged(4000×g, 5 minutes) and 500 μL of the supernatant was transferred to ashallow-well plate for analysis by HPLC.

Typical Procedure for Small-Scale Aziridination Bioconversions UnderAnaerobic Conditions Using Whole Cells and Purified Enzymes.

E. coli BL21(DE3) cells containing P450 or P411 enzymes were grown fromglycerol stock overnight (37° C., 250 rpm) in 5 ml Luria broth with 0.1mg mL⁻¹ ampicillin. The preculture was used to inoculate 45 mL ofHyperbroth medium (prepared from AthenaES© powder, 0.1 mg mL¹ampicillin) in a 125 mL Erlenmeyer flask; this culture was incubated at37° C., 220 rpm for 2 h and 30 min. After, the cultures were cooled onice and induced with 0.5 mM IPTG and 1 mM 5-aminolevulinic acid (finalconcentration). Expression was conducted at room temperature, 120 rpm,20 h. The cultures were then harvested and resuspended to OD₆₀₀=30 inM9-N. Aliquots of the cell suspension (4 mL) were used for determinationof the P450 or P411 expression level after lysis. E. coli cells(OD₆₀₀=30) were made anaerobic by sparging with argon in a sealed 6 mLcrimp vial for at least 30 minutes. To a 2 mL crimp vial was then addedglucose (250 mM in M9-N, 40 μL) and the GOX solution describedpreviously (20 μL). The headspace of the sealed 2 mL reaction vial wasmade anaerobic by flushing argon over the solution. Resuspended cells(320 μL), followed by olefin substrate (10 μL, 300 mM in DMSO), thentosyl azide (10 μL, 100 mM in DMSO) were added to 2 mL reaction vial viasyringe under continuous flow of argon. Final concentrations of reagentswere typically: 2.5 mM tosyl azide, 7.5 mM olefin, 25 mM glucose. The noenzyme control experiment was conducted using E. coli BL21 (DE3) cellscontaining empty pET22b(+) vector with the same reaction conditions asdescribed above. Purified enzyme reactions were conducted as describedpreviously, using 2.5 mM TsN₃ and 7.5 mM olefin (Farwell, C. C. et al.J. Am. Chem. Soc., 136, 8766-8771 (2014)). Sodium dithionite (5 mM) wasused as reductant for reactions with hemin, myoglobin, cytochrome C,CYP119, and P450_(Rhf). The reactions were shaken on a table-top shakeplate (40 rpm) at room temperature for 4 hours. The reactions werequenched by adding acetonitrile (460 μL) and the resulting mixture wastransferred to a microcentrifuge tube and centrifuged at 14,000 rpm for5 minutes. The solution (540 μL) was transferred to an HPLC vial,charged with internal standard (60 μL, 10 mM 1,3,5-trichlorobenzene inacetonitrile), and analyzed by HPLC.

Reactions for chiral HPLC analysis were performed on a 2 mL scale withthe same concentration of reagents and using a similar procedure asdescribed above. Briefly, cells containing P450 or P411 enzymes wereexpressed and resuspended to an OD₆₀₀=30 in M9-N, and then degassed bysparging with argon in a sealed 6 mL crimp vial for at least 30 minutes.To a 6 mL crimp vial was then added glucose (250 mM in M9-N, 200 μL) andthe GOX mixture described previously (100 μL). The headspace of thesealed 2 mL reaction vial was made anaerobic by flushing argon over thesolution. Resuspended cells (1600 μL), followed by olefin substrate (50μL, 300 mM in DMSO), then tosyl azide (50 μL, 100 mM in DMSO) were addedto 6 mL reaction vial via syringe under continuous flow of argon.Reactions were quenched with 2 mL acetonitrile, extracted with ethylacetate, dried and resuspended in acetone (200 μL), and purified by C18semi-preparative HPLC. The purified material was dried, resuspended inacetonitrile, and analyzed by SFC for enantioselectivity.

Determination of Initial Rates.

All initial rate experiments were conducted in an anaerobic chamber.Initial rate measurements were accomplished using 0.2 mol % purifiedenzymes in 400 μL scale reactions. A sealed 6-mL vial charged withglucose (250 mM, 480 μL), NADPH (100 mM, 480 μL), and potassiumphosphate buffer (0.1 M, pH=8.0, 3240 μL) was sparged for at least 30minutes with argon. After the degassing was complete, the reactionsolution, 2-mL vials charged with GOX solution (20 μL), and purifiedprotein (250 μM in potassium phosphate buffer), kept on ice, werebrought into the anaerobic chamber. The reaction solution (350 μL) wasadded to each 2-mL vial and allowed to equilibrate in the anaerobicchamber for 30 minutes. Reaction vials were then placed on a shaker (40rpm), charged with 10 μL purified protein (250 μM in potassium phosphatebuffer) and 4-methyl styrene substrate (10 μL, 300 mM in DMSO) followedby tosyl azide (10 μL, 100 mM in DMSO). Reactions were set up induplicate and products quantified at 1-2 minute intervals by quenchingwith acetonitrile (460 μL). The resulting mixture was removed from theanaerobic chamber, transferred to a microcentrifuge tube and centrifugedat 14,000 rpm for 5 minutes. The solution (540 μL) was transferred to anHPLC vial, charged with internal standard (60 μL, 10 mM1,3,5-trichlorobenzene in acetonitrile), and analyzed by HPLC. The ratesof aziridination and azide reduction for different enzyme variants arepresented in FIG. 6. The rate of azide reduction was determined in thepresence of olefin 3 (7.5 mM). Initial rates are plotted for individualenzymes in FIG. 7 A-C.

Assignment of Absolute Stereochemistry.

Absolute stereochemistry of enzymatically produced aziridine 6 wasassigned by chiral HPLC analysis and optical rotation. In particular,absolute stereochemistry of 6 was previously assigned by chiral HPLCusing Chiracel OJ column (isopropanol/n-hexane mobile phase), with (S)-6the earlier eluting enantiomer (Takeda Y., J. Am. Chem. Soc., 136,8544-7 (2014)). Analytically enantiopure 6 produced byP-I263F-A328V-L437V was subjected to the same chiral HPLC conditions andobserved to be the earlier eluting enantiomer (FIGS. 9A-B), leading toan assignment of (S)-6. Further support for this assignment came frommeasuring optical rotation. The optical rotation values for enantiomersof 7 have been previously reported (R)-6 [α_(D) ²⁵]−80.25 (c=0.8, CHCl₃)and (S)-6 [α_(D) ²⁰]+26.7 (c=0.7, CHCl₃) (Alonso, D. A., et al., J. Org.Chem., 63, 9455-9461 (1998); Wang, X. et al., Chem. Eur. J., 12,4568-4575 (2006)). Optical rotation measurement of analyticallyenantiopure 6 produced by P-I263F-A328V-L437V gave [α_(D) ²⁵]+80.2(c=1.2, CHCl₃), revealing it to be (S)-6. Similarly, the opticalrotation of P-I263F-A328V-L437V produced 4 (analytically enantiopure)was measured to be [α_(D) ²⁵]+106.1 (c=0.45, CHCl₃). By analogy, theconfiguration of enzymatically preferred (+)-4 is assigned as (S)-4.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

Informal Sequence Listing

CYP102A1 Cytochrome P450 (BM3) Bacillus megateriumGenBank Accession No. AAA87602 >gi|142798|gb|AAA87602.1|cytochrome P-450:NADPH-P-450 reductase  precursor [Bacillus megaterium]SEQ ID NO: 1 TIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIKEACDESRFDK NLSQALKFVR DFAGDGLFTS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMMVDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVRALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLNGKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLVDPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQLHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLKHFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHNTPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGHPPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIADRGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMHGAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFGLDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVELEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDEKQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLIMVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIITLHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYADVHQVSEADAR LWLQQLEEKG RYAKDVWAG CYP102A1B. megaterium >gi|281191140|gb|ADA57069.1|NADPH-cytochrome P450 reductase 102A1V9 [Bacillus megaterium]SEQ ID NO: 2MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYENLDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191138|gb|ADA57068.1|NADPH-cytochrome P450 reductase 102A1V10 [Bacillus megaterium]SEQ ID NO: 3MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAFEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETFAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFNLDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVATRFGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191126|gb|ADA57062.1|NADPH-cytochrome P450 reductase 102A1V4 [Bacillus megaterium]SEQ ID NO: 4MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEATRVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGEDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYENLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSERSTRHLEIALPKEASYQEGDHLGVIPRNYEGIVNRVTAREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAPFRSFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191124|gb|ADA57061.1|NADPH-cytochrome P450 reductase 102A1V8 [Bacillus megaterium]SEQ ID NO: 5MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPRVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYENLDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191120|gb|ADA57059.1|NADPH-cytochrome P450 reductase 102A1V3 [Bacillus megaterium]SEQ ID NO: 6MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYENLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQLGSERSTRHLEIALPKEASYQEGDHLGVIPRNYEGIVNRVTAREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSISPRYYSISSSPHVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVMERDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191118|gb|ADA57058.1|NADPH-cytochrome P450 reductase 102A1V7 [Bacillus megaterium]SEQ ID NO: 7MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPPEGAVLIVTASYNGHPPDNAKEFVDWLDQASADEVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYENLDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNEPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191112|gb|ADA57055.1|NADPH-cytochrome P450 reductase 102A1V2 [Bacillus megaterium]SEQ ID NO: 8MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEATRVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGEDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYENLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQLGSERSTRHLEIALPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSISPRYYSISSSPHVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVMERDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|269315992|gb|ACZ37122.1|cytochrome P450:NADPH P450 reductase  [Bacillus megaterium] SEQ ID NO: 9MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEVKGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYENLDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAGCYP102A1 B. megaterium >gi|281191116|gb|ADA57057.1|NADPH-cytochrome P450 reductase 102A1V6 [Bacillus megaterium]SEQ ID NO: 10MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQDDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVEGCGDKNWATTYQKVPAFIDETLSAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYENLNIENSEDNASTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTTREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLTKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFVSTPQSGFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVVEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHKVSEADARLWLQQLEEKSRYAKDVWAGCYP102A1 B. megaterium >gi|281191114|gb|ADA57056.1|NADPH-cytochrome P450 reductase 102A1V5 [Bacillus megaterium]SEQ ID NO: 11MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQDDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAFEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLSAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFNLNIENSEDNASTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTTRFGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLTKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFVSTPQSGFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVVEQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHKVSEADARLWLQQLEEKSRYAKDVWAGCYP153A6 Mycobacterium sp. HXN-1500GenBank Accession No.: CAH04396 >gi|51997117|emb|CAH04396.1|cytochrome P450 alkane hydroxylase [Mycobacterium sp. HXN-1500]SEQ ID NO: 12   1MTEMTVAASD ATNAAYGMAL EDIDVSNPVL FRDNTWHPYF KRLREEDPVH YCKSSMFGPY  61WSVTKYRDIM AVETNPKVFS SEAKSGGITI MDDNAAASLP MFIAMDPPKH DVQRKTVSPI 121VAPENLATME SVIRQRTADL LDGLPINEEF DWVHRVSIEL TTKMLATLFD FPWDDRAKLT 181RWSDVTTALP GGGIIDSEEQ RMAELMECAT YFTELWNQRV NAEPKNDLIS MMAHSESTRH 241MAPEEYLGNI VLLIVGGNDT TRNSMTGGVL ALNEFPDEYR KLSANPALIS SMVSEIIRWQ 301TPLSHMRRTA LEDIEFGGKH IRQGDKVVMW YVSGNRDPEA IDNPDTFIID RAKPRQHLSF 361GFGIHRCVGN RLAELQLNIL WEEILKRWPD PLQIQVLQEP TRVLSPFVKG YESLPVRINACYP5013C2 Tetrahymena thermophileGenBank Accession No.: ABY59989 >gi|164519863|gb|ABY59989.1|cytochrome P450 monooxygenase CYP5013C2 [Tetrahymena thermophila]SEQ ID NO: 13   1MIFELILIAV ALFAYFKIAK PYFSYLKYRK YGKGFYYPIL GEMIEQEQDL KQHADADYSV  61HHALDKDPDQ KLFVTNLGTK VKLRLIEPEI IKDFFSKSQY YQKDQTFIQN ITRFLKNGIV 121FSEGNTWKES RKLFSPAFHY EYIQKLTPLI NDITDTIFNL AVKNQELKNF DPIAQIQEIT 181GRVIIASFFG EVIEGEKFQG LTIIQCLSHI INTLGNQTYS IMYFLFGSKY FELGVTEEHR 241KFNKFIAEFN KYLLQKIDQQ IEIMSNELQT KGYIQNPCIL AQLISTHKID EITRNQLFQD 301FKTFYIAGMD TTGHLLGMTI YYVSQNKDIY TKLQSEIDSN TDQSAHGLIK NLPYLNAVIK 361ETLRYYGPGN ILFDRIAIKD HELAGIPIKK GTIVTPYAMS MQRNSKYYQD PHKYNPSRWL 421EKQSSDLHPD ANIPFSAGQR KCIGEQLALL EARIILNKFI KMFDFTCPQD YKLMMNYKFL 481SEPVNPLPLQ LTLRKQ Nonomuraea dietziae >gi|445067389|gb|AGE14547.1|cytochrome P450 hydroxylase sb8  [Nonomuraea dietziae]GenBank Accession No.: AGE14547 SEQ ID NO: 14VNIDLVDQDHYATEGPPHEQMRWLREHAPVYWHEGEPGFWAVTRHEDVVHVSRHSDLESSARRLALFNEMPEEQRELQRMMMLNQDPPEHTRRRSLVNRGFTPRTIRALEQHIRDICDDLLDQCSGEGDFVTDLAAPLPLYVICELLGAPVADRDKIFAWSNRMIGAQDPDYAASPEEGGAAAMEVYAYASELAAQRRAAPRDDIVTKLLQSDENGESLTENEFELFVLLLVVAGNETTRNAASGGMLTLFEHPDQWDRLVADPSLAATAADEIVRWVSPVNLFRRTATADLTLGGQQVKADDKVVVFYSSANRDASVESDPEVEDIGRSPNPHIGEGGGGAHFCLGNHLAKLELRVLFEQLARREPRMRQTGEARRLRSNFINGIKTLPVTLG CYP2R1 Homo sapiensGenBank Accession No.: NP 078790 >gi|45267826|ref|NP_078790.2|vitamin D 25-hydroxylase [Homo sapiens] SEQ ID NO: 15   1MWKLWRAEEG AAALGGALFL LLFALGVRQL LKQRRPMGFP PGPPGLPFIG NIYSLAASSE  61LPHVYMRKQS QVYGEIFSLD LGGISTVVLN GYDVVKECLV HQSEIFADRP CLPLFMKMTK 121MGGLLNSRYG RGWVDHRRLA VNSFRYFGYG QKSFESKILE ETKFFNDAIE TYKGRPFDFK 181QLITNAVSNI TNLIIFGERF TYEDTDFQHM IELFSENVEL AASASVFLYN AFPWIGILPF 241GKHQQLFRNA AVVYDFLSRL IEKASVNRKP QLPQHFVDAY LDEMDQGKND PSSTFSKENL 301IFSVGELIIA GTETTTNVLR WAILFMALYP NIQGQVQKEI DLIMGPNGKP SWDDKCKMPY 361TEAVLHEVLR FCNIVPLGIF HATSEDAVVR GYSIPKGTTV ITNLYSVHFD EKYWRDPEVF 421HPERFLDSSG YFAKKEALVP FSLGRRHCLG EHLARMEMFL FFTALLQRFH LHFPHELVPD 481LKPRLGMTLQ PQPYLICAER R CYP2R1 Macca mulattaGenBank Accession No.: NP 001180887 >gi|302565346|ref|NP_001180887.1|vitamin D 25-hydroxylase  [Macaca mulatta] SEQ ID NO: 16   1MWKLWGGEEG AAALGGALFL LLFALGVRQL LKLRRPMGFP PGPPGLPFIG NIYSLAASAE  61LPHVYMRKQS QVYGEIFSLD LGGISTVVLN GYDVVKECLV HQSGIFADRP CLPLFMKMTK 121MGGLLNSRYG QGWVEHRRLA VNSFRYFGYG QKSFESKILE ETKFFTDAIE TYKGRPFDFK 181QLITSAVSNI TNLIIFGERF TYEDTDFQHM IELFSENVEL AASASVFLYN AFPWIGILPF 241GKHQQLFRNA SVVYDFLSRL IEKASVNRKP QLPQHFVDAY FDEMDQGKND PSSTFSKENL 301IFSVGELIIA GTETTTNVLR WAILFMALYP NIQGQVQKEI DLIMGPNGKP SWDDKFKMPY 361TEAVLHEVLR FCNIVPLGIF HATSEDAVVR GYSIPKGTTV ITNLYSVHFD EKYWRDPEVF 421HPERFLDSSG YFAKKEALVP FSLGRRHCLG EQLARMEMFL FFTALLQRFH LHFPHELVPD 481LKPRLGMTLQ PQPYLICAER R CYP2R1 Canis familiarisGenBank Accession No.: XP_854533 >gi|73988871|ref|XP_854533.11 PREDICTED: vitamin D 25-hydroxylase [Canis lupus familiaris] SEQ ID NO: 17   1MRGPPGAEAC AAGLGAALLL LLFVLGVRQL LKQRRPAGFP PGPSGLPFIG NIYSLAASGE  61LAHVYMRKQS RVYGEIFSLD LGGISAVVLN GYDVVKECLV HQSEIFADRP CLPLFMKMTK 121MGGLLNSRYG RGWVDHRKLA VNSFRCFGYG QKSFESKILE ETNFFIDAIE TYKGRPFDLK 181QLITNAVSNI TNLIIFGERF TYEDTDFQHM IELFSENVEL AASASVFLYN AFPWIGIIPF 241GKHQQLFRNA AVVYDFLSRL IEKASINRKP QSPQHFVDAY LNEMDQGKND PSCTFSKENL 301IFSVGELIIA GTETTTNVLR WAILFMALYP NIQGQVQKEI DLIMGPTGKP SWDDKCKMPY 361TEAVLHEVLR FCNIVPLGIF HATSEDAVVR GYSIPKGTTV ITNLYSVHFD EKYWRNPEIF 421YPERFLDSSG YFAKKEALVP FSLGKRHCLG EQLARMEMFL FFTALLQRFH LHFPHGLVPD 481LKPRLGMTLQ PQPYLICAER R CYP2R1 Mus musculusGenBank Accession No.: AAI08963 >gi|80477959|gb|AAI08963.1|Cyp2r1 protein [Mus musculus] SEQ ID NO: 18   1MGDEMDQGQN DPLSTFSKEN LIFSVGELII AGTETTTNVL RWAILFMALY PNIQGQVHKE  61IDLIVGHNRR PSWEYKCKMP YTEAVLHEVL RFCNIVPLGI FHATSEDAVV RGYSIPKGTT 121VITNLYSVHF DEKYWKDPDM FYPERFLDSN GYFTKKEALI PFSLGRRHCL GEQLARMEMF 181LFFTSLLQQF HLHFPHELVP NLKPRLGMTL QPQPYLICAE RR CYP152A6Bacillus halodurans C-125GenBank Accession No.: NP|242623 >gi|15614320|ref|NP|242623.11 fatty acid alpha hydroxylase [Bacillus halodurans C-125] SEQ ID NO: 19   1MKSNDPIPKD SPLDHTMNLM REGYEFLSHR MERFQTDLFE TRVMGQKVLC IRGAEAVKLF  61YDPERFKRHR ATPKRIQKSL FGENAIQTMD DKAHLHRKQL FLSMMKPEDE QELARLTHET 121WRRVAEGWKK SRPIVLFDEA KRVLCQVACE WAEVPLKSTE IDRRAEDFHA MVDAFGAVGP 181RHWRGRKGRR RTERWIQSII HQVRTGSLQA REGSPLYKVS YHRELNGKLL DERMAAIELI 241NVLRPIVAIA TFISFAAIAL QEHPEWQERL KNGSNEEFHM FVQEVRRYYP FAPLIGAKVR 301KSFTWKGVRF KKGRLVFLDM YGTNHDPKLW DEPDAFRPER FQERKDSLYD FIPQGGGDPT 361KGHRCPGEGI TVEVMKTTMD FLVNDIDYDV PDQDISYSLS RMPTRPESGY IMANIERKYE 421 HAaryC Streptomyces parvusGenBank Accession No.: AFM80022 >gi|392601346|gb|AFM80022.1|cytochrome P450 +Streptomyces parvus+ SEQ ID NO: 20   1MYLGGRRGTE AVGESREPGV WEVFRYDEAV QVLGDHRTFS SDMNHFIPEE QRQLARAARG  61NFVGIDPPDH TQLRGLVSQA FSPRVTAALE PRIGRLAEQL LDDIVAERGD KASCDLVGEF 121AGPLSAIVIA ELFGIPESDH TMIAEWAKAL LGSRPAGELS IADEAAMQNT ADLVRRAGEY 181LVHHITERRA RPQDDLTSRL ATTEVDGKRL DDEEIVGVIG MFLIAGYLPA SVLTANTVMA 241LDEHPAALAE VRSDPALLPG AIEEVLRWRP PLVRDQRLTT RDADLGGRTV PAGSMVCVWL 301ASAHRDPFRF ENPDLFDIHR NAGRHLAFGK GIHYCLGAPL ARLEARIAVE TLLRRFERIE 361IPRDESVEFH ESIGVLGPVR LPTTLFARR CYP101A1 Pseudomonas putidaUniprot Accession No.: P00183 >sp|P00183|CPXA_PSEPU Camphor 5-monooxygenase OS =Pseudomonas putida  GN = camC PE = 1 SV = 2 SEQ ID NO: 21TTETIQSNANLAPLPPHVPEHLVEDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYEDYRHESSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEELLRRFSLVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV Homo sapiens CYP2D7GenBank Accession No.: AA049806 >gi|37901459|gb|AA049806.1|cytochrome P450 [Homo sapiens] SEQ ID NO: 22GLEALVPLA MIVAIFLLLV DLMHRHQRWA ARYPPGPLPL PGLGNLLHVD FQNTPYCFDQLRRRFGDVFN LQLAWTPVVV LNGLAAVREA MVTRGEDTAD RPPAPIYQVL GFGPRSQGVILSRYGPAWRE QRRFSVSTLR NLGLGKKSLE QWVTEEAACL CAAFADQAGR PFRPNGLLDKAVSNVIASLT CGRRFEYDDP RFLRLLDLAQ EGLKEESGFL REVLNAVPVL PHIPALAGKVLRFQKAFLTQ LDELLTEHRM TWDPAQPPRD LTEAFLAKKE KAKGSPESSF NDENLRIVVGNLFLAGMVTT LTTLAWGLLL MILHLDVQRG RRVSPGCSPI VGTHVCPVRV QQEIDDVIGQVRRPEMGDQV HMPYTTAVIH EVQRFGDIVP LGVTHMTSRD IEVQGFRIPK GTTLITNLSSVLKDEAVWEK PFRFHPEHFL DAQGHFVKPE AFLPFSAGRR ACLGEPLARM ELFLFFTSLLQHFSFSVAAG QPRPSHSRVV SFLVTPSPYE LCAVPR Rattus norvegicus CYPC27GenBank Accession No.: AAB02287 >gi|1374714|gb|AAB02287.1|cytochrome P450 [Rattus norvegicus] SEQ ID NO: 23AVLSRMRLRWALLDTRVMGHGLCPQGARAKAAIPAALRDHESTEGPGTGQDRPRLRSLAELPGPGTLRFLFQLFLRGYVLHLHELQALNKAKYGPMWTTTEGTRTNVNLASAPLLEQVMRQEGKYPIRDSMEQWKEHRDHKGLSYGIFITQGQQWYHLRHSLNQRMLKPAEAALYTDALNEVISDFIARLDQVRTESASGDQVPDVAHLLYHLALEAICYILFEKRVGCLEPSIPEDTATFIRSVGLMEKNSVYVTFLPKWSRPLLPFWKRYMNNWDNIFSFGEKMIHQKVQEIEAQLQAAGPDGVQVSGYLHFLLTKELLSPQETVGTFPELILAGVDTTSNTLTWALYHLSKNPEIQEALHKEVTGVVPFGKVPQNKDFAHMPLLKAVIKETLRLYPVVPTNSRIITEKETEINGFLFPKNTQFVLCTYVVSRDPSVFPEPESFQPHRWLRKREDDNSGIQHPFGSVPFGYGVRSCLGRRIAELEMQLLLSRLIQKYEVVLSPGMGEVKSVSRIVLVPSKKVSLRFLQRQ CYP2B4 Oryctolagus cuniculusGenBank Accession No. AAA65840 >gi|164959|gb|AAA65840.1|cytochrome P-450 [Oryctolagus cuniculus] SEQ ID NO: 24MEFSLLLLLAFLAGLLLLLFRGHPKAHGRLPPGPSPLPVLGNLLQMDRKGLLRSFLRLREKYGDVFTVYLGSRPVVVLCGTDAIREALVDQAEAFSGRGKIAVVDPIFQGYGVIFANGERWRALRRFSLATMRDFGMGKRSVEERIQEEARCLVEELRKSKGALLDNTLLFHSITSNIICSIVEGKREDYKDPVFLRLLDLFFQSFSLISSFSSQVFELFPGFLKHFPGTHRQIYRNLQEINTFIGQSVEKHRATLDPSNPRDFIDVYLLRMEKDKSDPSSEFHHQNLILTVLSLFFAGTETTSTTLRYGELLMLKYPHVTERVQKEIEQVIGSHRPPALDDRAKMPYTDAVIHEIQRLGDLIPFGVPHTVTKDTQFRGYVIPKNTEVFPVLSSALHDPRYFETPNTENPGHFLDANGALKRNEGFMPFSLGKRICLGEGIARTELFLFFTTILQNFSIASPVPPEDIDLTPRESGVGNV PPSYQIRFLARCYP102A2 Bacillus subtilisUniprot Accession No. 008394 >sp|O08394|CYPD_BACSU Probable bifunctional P-450/NADPH-P450 reductase 1 OS = Bacillus subtilis (strain 168) GN = cypD  PE = 3 SV = 1SEQ ID NO: 25MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGALEKVRAFSGDGLFTSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLIQKWARLNPNEAVDVPGDMTRLTLDTIGLCGFNYRFNSYYRETPHPFINSMVRALDEAMHQMQRLDVQDKLMVRTKRQFRHDIQTMESLVDSIIAERRANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFATYFLLKHPDKLKKAYEEVDRVLTDAAPTYKQVLELTYIRMILNESLRLWPTAPAFSLYPKEDTVIGGKFPITTNDRISVLIPQLHRDRDAWGKDAEEFRPERFEHQDQVPHHAYKPFGNGQRACIGMQFALHEATLVLGMILKYFTLIDHENYELDIKQTLTLKPGDFHIRVQSRNQDAIHADVQAVEKAASDEQKEKTEAKGTSVIGLNNRPLLVLYGSDTGTAEGVARELADTASLHGVRTETAPLNDRIGKLPKEGAVVIVTSSYNGKPPSNAGQFVQWLQEIKPGELEGVHYAVEGCGDHNWASTYQYVPRFIDEQLAEKGATRFSARGEGDVSGDFEGQLDEWKKSMWADAIKAFGLELNENADKERSTLSLQFVRGLGESPLARSYEASHASIAENRELQSADSDRSTRHIEIALPPDVEYQEGDHLGVLPKNSQTNVSRILHREGLKGTDQVTLSASGRSAGHLPLGRPVSLHDLLSYSVEVQEAATRAQIRELAAFTVCPPHRRELEELSAEGVYQEQILKKRISMLDLLEKYEACDMPFERFLELLRPLKPRYYSISSSPRVNPRQASITVGVVRGPAWSGRGEYRGVASNDLAERQAGDDVVMFIRTPESRFQLPKDPETPIIMVGPGTGVAPERGELQARDVLKREGKTLGEAHLYFGCRNDRDFIYRDELERFEKDGIVTVHTAFSRKEGMPKTYVQHLMADQADTLISILDRGGRLYVCGDGSKMAPDVEAALQKAYQAVHGTGEQEAQNWLRHLQDTGMYAKDVWAGI CYP102A3 Bacillus subtilisUniprot Accession No.008336 >sp|008336|CYPE_BACSU Probable bifunctional P-450/NADPH-P450 reductase 2 OS = Bacillus subtilis (strain 168) GN = cypE  PE = 2 SV = 1SEQ ID NO: 26MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFREDFPGVSSVFVSGHNLVAEVCDESREDKNLGKGLQKVREFGGDGLFTSWTHEPNWQKAHRILLPSFSQKAMKGYHSMMLDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDSQHPFITSMLRALKEAMNQSKRLGLQDKMMVKTKLQFQKDIEVMNSLVDRMIAERKANPDDNIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYKQIQQLKYTRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQADIKAETKPKETKPKHGTPLLVLYGSNLGTAEGIAGELAAQGRQMGETAETAPLDDYIGKLPEEGAVVIVTASYNGSPPDNAAGFVEWLKELEEGQLKGVSYAVEGCGNRSWASTYQRIPRLIDDMMKAKGASRLTE IGEGDAADDFESHRESWENRFWKETMDAFDINEIAQKEDRPSLSIAFLSEATETPVAKAYGAFEGVVLENRELQTADSTRSTRHIELEIPAGKTYKEGDHIGIMPKNSRELVQRVLSREGLQSNHVIKVSGSAHMSHLPMDRPIKVADLLSSYVELQEPASRLQLRELASYTVCPPHQKELEQLVLDDGIYKEQVLAKRLTMLDFLEDYPACEMPFERFLALLPSLKPRYYSISSSPKVHANIVSMTVGVVKASAWSGRGEYRGVASNYLAELNTGDAAACFIRTPQSGFQMPDEPETPMIMVGPGTGIAPERGFIQARSVLKKEGSTLGEALLYFGCRRPDHDDLYREELDQAEQEGLVTIRRCYSRVENESKGYVQHLLKQDSQKLMTLIEKGAHIYVCGDGSQMAPDVEKTLRWAYETEKGASQEESADWLQKLQDQKRYIKDVWTGN CYP102A1 B. megaterium DSM 32Uniprot Accession No. P14779>sp|P14779|CPXB_BACME Bifunctional P-450/NADPH-P450 reductase OS+32Bacillusmegaterium GN = cyp102A1 PE = 1 SV = 2 SEQ ID NO: 27    1MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK   61EACDESRFDK NLSQALKFVR DFAGDGLFTS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM  121VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR  181ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN  241GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV  301DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ  361LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK  421HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN  481TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH  541PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD  601RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH  661GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG  721LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE  781LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE  841KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI  901MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT  961LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 1021VHQVSEADAR LWLQQLEEKG RYAKDVWAG CYP102A5 B. cereus ATCC14579GenBank Accession No. AAP10153 >gi|29896875|gb|AAP10153.1|NADPH-cytochrome P450 reductase  [Bacillus cereus ATCC 14579]SEQ ID NO: 28    1MEKKVSAIPQ PKTYGPLGNL PLIDKDKPTL SFIKIAEEYG PIFQIQTLSD TIIVVSGHEL   61VAEVCDETRF DKSIEGALAK VRAFAGDGLF TSETHEPNWK KAHNILMPTF SQRAMKDYHA  121MMVDIAVQLV QKWARLNPNE NVDVPEDMTR LTLDTIGLCG FNYRFNSFYR ETPHPFITSM  181TRALDEAMHQ LQRLDIEDKL MWRTKRQFQH DIQSMFSLVD NIIAERKSSG DQEENDLLSR  241MLNVPDPETG EKLDDENIRF QIITFLIAGH ETTSGLLSFA IYFLLKNPDK LKKAYEEVDR  301VLTDPTPTYQ QVMKLKYMRM ILNESLRLWP TAPAFSLYAK EDTVIGGKYP IKKGEDRISV  361LIPQLHRDKD AWGDNVEEFQ PERFEELDKV PHHAYKPFGN GQRACIGMQF ALHEATLVMG  421MLLQHFELID YQNYQLDVKQ TLTLKPGDFK IRILPRKQTI SHPTVLAPTE DKLKNDEIKQ  481HVQKTPSIIG ADNLSLLVLY GSDTGVAEGI ARELADTASL EGVQTEVVAL NDRIGSLPKE  541GAVLIVTSSY NGKPPSNAGQ FVQWLEELKP DELKGVQYAV FGCGDHNWAS TYQRIPRYID  601EQMAQKGATR FSKRGEADAS GDFEEQLEQW KQNMWSDAMK AFGLELNKNM EKERSTLSLQ  661FVSRLGGSPL ARTYEAVYAS ILENRELQSS SSDRSTRHIE VSLPEGATYK EGDHLGVLPV  721NSEKNINRIL KRFGLNGKDQ VILSASGRSI NHIPLDSPVS LLALLSYSVE VQEAATRAQI  781REMVTFTACP PHKKELEALL EEGVYHEQIL KKRISMLDLL EKYEACEIRF ERFLELLPAL  841KPRYYSISSS PLVAHNRLSI TVGVVNAPAW SGEGTYEGVA SNYLAQRHNK DEIICFIRTP  901QSNFELPKDP ETPIIMVGPG TGIAPFRGFL QARRVQKQKG MNLGQAHLYF GCRHPEKDYL  961YRTELENDER DGLISLHTAF SRLEGHPKTY VQHLIKQDRI NLISLLDNGA HLYICGDGSK 1021MAPDVEDTLC QAYQEIHEVS EQEARNWLDR VQDEGRYGKD VWAGI CYP102A7B. licheniformis ATTC1458GenBank Accession No. YP 079990 >gi|52081199|ref|YP_079990.11 cytochrome P450/NADPH-ferrihemoproteinreductase [Bacillus licheniformis DSM 13 = ATCC 14580] SEQ ID NO: 29   1 MNKLDGIPIP KTYGPLGNLP LLDKNRVSQS LWKIADEMGP IFQFKFADAI GVFVSSHELV  61 KEVSEESRFD KNMGKGLLKV REFSGDGLFT SWTEEPNWRK AHNILLPSFS QKAMKGYHPM 121 MQDIAVQLIQ KWSRLNQDES IDVPDDMTRL TLDTIGLCGF NYRFNSFYRE GQHPFIESMV 181 RGLSEAMRQT KRFPLQDKLM IQTKRRFNSD VESMFSLVDR IIADRKQAES ESGNDLLSLM 241 LHAKDPETGE KLDDENIRYQ IITFLIAGHE TTSGLLSFAI YLLLKHPDKL KKAYEEADRV 301 LTDPVPSYKQ VQQLKYIRMI LNESIRLWPT APAFSLYAKE ETVIGGKYLI PKGQSVTVLI 361 PKLHRDQSVW GEDAEAFRPE RFEQMDSIPA HAYKPFGNGQ RACIGMQFAL HEATLVLGMI 421 LQYFDLEDHA NYQLKIKESL TLKPDGFTIR VRPRKKEAMT AMPGAQPEEN GRQEERPSAP 481 AAENTHGTPL LVLYGSNLGT AEEIAKELAE EAREQGFHSR TAELDQYAGA IPAEGAVIIV 541 TASYNGNPPD CAKEFVNWLE HDQTDDLRGV KYAVFGCGNR SWASTYQRIP RLIDSVLEKK 601 GAQRLHKLGE GDAGDDFEGQ FESWKYDLWP LLRTEFSLAE PEPNQTETDR QALSVEFVNA 661 PAASPLAKAY QVFTAKISAN RELQCEKSGR STRHIEISLP EGAAYQEGDH LGVLPQNSEV 721 LIGRVFQRFG LNGNEQILIS GRNQASHLPL ERPVHVKDLF QHCVELQEPA TRAQIRELAA 781 HTVCPPHQRE LEDLLKDDVY KDQVLNKRLT MLDLLEQYPA CELPFARFLA LLPPLKPRYY 841 SISSSPQLNP RQTSITVSVV SGPALSGRGH YKGVASNYLA GLEPGDAISC FIREPQSGFR 901 LPEDPETPVI MVGPGTGIAP YRGFLQARRI QRDAGVKLGE AHLYFGCRRP NEDFLYRDEL 961 EQAEKDGIVH LHTAFSRLEG RPKTYVQDLL REDAALLIHL LNEGGRLYVC GDGSRMAPAV1021 EQALCEAYRI VQGASREESQ SWLSALLEEG RYAKDVWDGG VSQHNVKADC IART CYPXB. thuringiensis serovar konkukian str.97-27GenBank Accession No. YP 037304 >gi|49480099|ref|YP_037304.11 NADPH-cytochrome P450 reductase [Bacillus thuringiensis serovar konkukian str. 97-27] SEQ ID NO: 30    1MDKKVSAIPQ PKTYGPLGNL PLIDKDKPTL SFIKLAEEYG PIFQIQTLSD TIIVVSGHEL   61VAEVCDETRF DKSIEGALAK VRAFAGDGLF TSETDEPNWK KAHNILMPTF SQRAMKDYHA  121MMVDIAVQLV QKWARLNPNE NVDVPEDMTR LTLDTIGLCG FNYRFNSFYR ETPHPFITSM  181TRALDEAMHQ LQRLDIEDKL MWRTKRQFQH DIQSMFSLVD NIIAERKSSE NQEENDLLSR  241MLNVQDPETG EKLDDENIRF QIITFLIAGH ETTSGLLSFA IYFLLKNPDK LKKAYEEVDR  301VLTDSTPTYQ QVMKLKYIRM ILNESLRLWP TAPAFSLYAK EDTVIGGKYP IKKGEDRISV  361LIPQLHRDKD AWGDDVEEFQ PERFEELDKV PHHAYKPFGN GQRACIGMQF ALHEATLVMG  421MLLQHFEFID YEDYQLDVKQ TLTLKPGDFK IRIVPRNQTI SHTTVLAPTE EKLKKHEIKK  481QVQKTPSIIG ADNLSLLVLY GSDTGVAEGI ARELADTASL EGVQTEVVAL NDRIGSLPKE  541GAVLIVTSSY NGKPPSNAGQ FVQWLEELKP DELKGVQYAV FGCGDHNWAS TYQRIPRYID  601EQMAQKGATR FSTRGEADAS GDFEEQLEQW KQSMWSDAMK AFGLELNKNM EKERSTLSLQ  661FVSRLGGSPL ARTYEAVYAS ILENRELQSS SSERSTRHIE ISLPEGATYK EGDHLGVLPI  721NNEKNVNRIL KRFGLNGKDQ VILSASGRSV NHIPLDSPVR LYDLLSYSVE VQEAATRAQI  781REMVTFTACP PHKKELESLL EDGVYQEQIL KKRISMLDLL EKYEACEIRF ERFLELLPAL  841KPRYYSISSS PLVAQDRLSI TVGVVNAPAW SGEGTYEGVA SNYLAQRHNK DEIICFIRTP  901QSNFQLPENP ETPIIMVGPG TGIAPFRGFL QARRVQKQKG MKVGEAHLYF GCRHPEKDYL  961YRTELENDER DGLISLHTAF SRLEGHPKTY VQHVIKEDRI HLISLLDNGA HLYICGDGSK 1021MAPDVEDTLC QAYQEIHEVS EQEARNWLDR LQEEGRYGKD VWAGI CYP102E1R. metallidurans CH34GenBank Accession No. YP 585608 >gi|94312398|ref|YP_585608.1|putative bifunctional P-450:NADPH-P450reductase 2 [Cupriavidus metallidurans CH34] SEQ ID NO: 31    1MSTATPAAAL EPIPRDPGWP IFGNLFQITP GEVGQHLLAR SRHHDGIFEL DFAGKRVPFV   61SSVALASELC DATRFRKIIG PPLSYLRDMA GDGLFTAHSD EPNWGCAHRI LMPAFSQRAM  121KAYFDVMLRV ANRLVDKWDR QGPDADIAVA DDMTRLTLDT IALAGFGYDF ASFASDELDP  181FVMAMVGALG EAMQKLTRLP IQDRFMGRAH RQAAEDIAYM RNLVDDVIRQ RRVSPTSGMD  241LLNLMLEARD PETDRRLDDA NIRNQVITFL IAGHETTSGL LTFALYELLR NPGVLAQAYA  301EVDTVLPGDA LPVYADLARM PVLDRVLKET LRLWPTAPAF AVAPFDDVVL GGRYRLRKDR  361RISVVLTALH RDPKVWANPE RFDIDRFLPE NEAKLPAHAY MPFGQGERAC IGRQFALTEA  421KLALALMLRN FAFQDPHDYQ FRLKETLTIK PDQFVLRVRR RRPHERFVTR QASQAVADAA  481QTDVRGHGQA MTVLCASSLG TARELAEQIH AGAIAAGFDA KLADLDDAVG VLPTSGLVVV  541VAATYNGRAP DSARKFEAML DADDASGYRA NGMRLALLGC GNSQWATYQA FPRRVFDFFI  601TAGAVPLLPR GEADGNGDFD QAAERWLAQL WQALQADGAG TGGLGVDVQV RSMAAIRAET  661LPAGTQAFTV LSNDELVGDP SGLWDFSIEA PRTSTRDIRL QLPPGITYRT GDHIAVWPQN  721DAQLVSELCE RLDLDPDAQA TISAPHGMGR GLPIDQALPV RQLLTHFIEL QDVVSRQTLR  781ALAQATRCPF TKQSIEQLAS DDAEHGYATK VVARRLGILD VLVEHPAIAL TLQELLACTV  841PMRPRLYSIA SSPLVSPDVA TLLVGTVCAP ALSGRGQFRG VASTWLQHLP PGARVSASIR  901TPNPPFAPDP DPAAPMLLIG PGTGIAPFRG FLEERALRKM AGNAVTPAQL YFGCRHPQHD  961WLYREDIERW AGQGVVEVHP AYSVVPDAPR YVQDLLWQRR EQVWAQVRDG ATIYVCGDGR 1021RMAPAVRQTL IEIGMAQGGM TDKAASDWFG GLVAQGRYRQ DVFN CYP505XA. fumigatus Af293GenBank Accession No. EAL92660 >gi|66852335|gb|EAL92660.1|P450 family fatty acid hydroxylase, putative[Aspergillus fumigatus Af293] SEQ ID NO: 32    1MSESKTVPIP GPRGVPLLGN IYDIEQEVPL RSINLMADQY GPIYRLTTFG WSRVFVSTHE   61LVDEVCDEER FTKVVTAGLN QIRNGVHDGL FTANFPGEEN WAIAHRVLVP AFGPLSIRGM  121FDEMYDIATQ LVMKWARHGP TVPIMVTDDF TRLTLDTIAL CAMGTRFNSF YHEEMHPFVE  181AMVGLLQGSG DRARRPALLN NLPTSENSKY WDDIAFLRNL AQELVEARRK NPEDKKDLLN  241ALILGRDPKT GKGLTDESII DNMITFLIAG HETTSGLLSF LFYYLLKTPN AYKKAQEEVD  301SVVGRRKITV EDMSRLPYLN AVMRETLRLR STAPLIAVHA HPEKNKEDPV TLGGGKYVLN  361KDEPIVIILD KLHRDPQVYG PDAEEFKPER MLDENFEKLP KNAWKPFGNG MRACIGRPFA  421WQEALLVVAI LLQNFNFQMD DPSYNLHIKQ TLTIKPKDFH MRATLRHGLD ATKLGIALSG  481SADRAPPESS GAASRVRKQA TPPAGQLKPM HIFFGSNTGT CETFARRLAD DAVGYGFAAD  541VQSLDSAMQN VPKDEPVVFI TASYEGQPPD NAAHFFEWLS ALKENELEGV NYAVFGCGHH  601DWQATFHRIP KAVNQLVAEH GGNRLCDLGL ADAANSDMFT DFDSWGESTF WPAITSKFGG  661GKSDEPKPSS SLQVEVSTGM RASTLGLQLQ EGLVIDNQLL SAPDVPAKRM IRFKLPSDMS  721YRCGDYLAVL PVNPTSVVRR AIRRFDLPWD AMLTIRKPSQ APKGSTSIPL DTPISAFELL  781STYVELSQPA SKRDLTALAD AAITDADAQA ELRYLASSPT RFTEEIVKKR MSPLDLLIRY  841PSIKLPVGDF LAMLPPMRVR QYSISSSPLA DPSECSITFS VLNAPALAAA SLPPAERAEA  901EQYMGVASTY LSELKPGERA HIAVRPSHSG FKPPMDLKAP MIMACAGSGL APFRGFIMDR  961AEKIRGRRSS VGADGQLPEV EQPAKAILYV GCRTKGKDDI HATELAEWAQ LGAVDVRWAY 1021SRPEDGSKGR HVQDLMLEDR EELVSLFDQG ARIYVCGSTG VGNGVRQACK DIYLERRRQL 1081RQAARERGEE VPAEEDEDAA AEQFLDNLRT KERYATDVFT CYP505A8 A. nidulans FGSC A4GenBank Accession No. EAA58234 >gi|40739044|gb|EAA58234.1|hypothetical protein AN6835.2  [Aspergillus nidulans FGSC A4]SEQ ID NO: 33    1MAEIPEPKGL PLIGNIGTID QEFPLGSMVA LAEEHGEIYR LRFPGRTVVV VSTHALVNET   61CDEKRFRKSV NSALAHVREG VHDGLFTAKM GEVNWEIAHR VLMPAFGPLS IRGMFDEMHD  121IASQLALKWA RYGPDCPIMV TDDFTRLTLD TLALCSMGYR FNSYYSPVLH PFIEAMGDFL  181TEAGEKPRRP PLPAVFFRNR DQKFQDDIAV LRDTAQGVLQ ARKEGKSDRN DLLSAMLRGV  241DSQTGQKMTD ESIMDNLITF LIAGHETTSG LLSFVFYQLL KHPETYRTAQ QEVDNVVGQG  301VIEVSHLSKL PYINSVLRET LRLNATIPLF TVEAFEDTLL AGKYPVKAGE TIVNLLAKSH  361LDPEVYGEDA LEFKPERMSD ELFNARLKQF PSAWKPFGNG MRACIGRPFA WQEALLVMAM  421LLQNFDFSLA DPNYDLKFKQ TLTIKPKDMF MKARLRHGLT PTTLERRLAG LAVESATQDK  481IVTNPADNSV TGTRLTILYG SNSGTCETLA RRIAADAPSK GFHVMRFDGL DSGRSALPTD  541HPVVIVTSSY EGQPPENAKQ FVSWLEELEQ QNESLQLKGV DFAVFGCFKE WAQTFHRIPK  601LVDSLLEKLG GSRLTDLGLA DVSTDELFST FETWADDVLW PRLVAQYGAD GKTQAHGSSA  661GHEAASNAAV EVTVSNSRTQ ALRQDVGQAM VVETRLLTAE SEKERRKKHL EIRLPDGVSY  721TAGDYLAVLP INPPETVRRA MRQFKLSWDA QITIAPSGPT TALPTDGPIA ANDIFSTYVE  781LSQPATRKDL RIMADATTDP DVQKILRTYA NETYTAEILT KSISVLDILE QHPAIDLPLG  841TFLLMLPSMR MRQYSISSSP LLTPTTATIT ISVLDAPSRS RSNGSRHLGV ATSYLDSLSV  901GDHLQVTVRK NPSSGFRLPS EPETTPMICI AAGSGIAPFR AFLQERAVMM EQDKDRKLAP  961ALLFFGCRAP GIDDLYREQL EEWQARGVVD ARWAFSRQSD DTKGCRHVDD RILADREDVV 1021KLWRDGARVY VCGSGALAQS VRSAMVTVLR DEMETTGDGS DNGKAEKWFD EQRNVRYVMD 1081VFD CYP505A3 A. oryzae ATCC42149Uniprot Accession No. Q2U4F1 >gi|121928062|sp|Q2U4F1|Q2U4F1_ASPOR Cytochrome P450SEQ ID NO: 34    1MRQNDNEKQI CPIPGPQGLP FLGNILDIDL DNGTMSTLKI AKTYYPIFKF TFAGETSIVI   61NSVALLSELC DETRFHKHVS FGLELLRSGT HDGLFTAYDH EKNWELAHRL LVPAFGPLRI  121REMFPQMHDI AQQLCLKWQR YGPRRPLNLV DDFTRTTLDT IALCAMGYRF NSFYSEGDFH  181PFIKSMVRFL KEAETQATLP SFISNLRVRA KRRTQLDIDL MRTVCREIVT ERRQTNLDHK  241NDLLDTMLTS RDSLSGDALS DESIIDNILT FLVAGHETTS GLLSFAVYYL LTTPDAMAKA  301AHEVDDVVGD QELTIEHLSM LKYLNAILRE TLRLMPTAPG FSVTPYKPEI IGGKYEVKPG  361DSLDVFLAAV HRDPAVYGSD ADEFRPERMS DEHFQKLPAN SWKPFGNGKR SCIGRAFAWQ  421EALMILALIL QSFSLNLVDR GYTLKLKESL TIKPDNLWAY ATPRPGRNVL HTRLALQTNS  481THPEGLMSLK HETVESQPAT ILYGSNSGTC EALAHRLAIE MSSKGRFVCK VQPMDAIEHR  541RLPRGQPVII ITGSYDGRPP ENARHFVKWL QSLKGNDLEG IQYAVFGCGL PGHHDWSTTF  601YKIPTLIDTI MAEHGGARLA PRGSADTAED DPFAELESWS ERSVWPGLEA AFDLVRHNSS  661DGTGKSTRIT IRSPYTLRAA HETAVVHQVR VLTSAETTKK VHVELALPDT INYRPGDHLA  721ILPLNSRQSV QRVLSLFQIG SDTILYMTSS SATSLPTDTP ISAHDLLSGY VELNQVATPT  781SLRSLAAKAT DEKTAEYLEA LATDRYTTEV RGNHLSLLDI LESYSVPSIE IQHYIQMLPL  841LRPRQYTISS SPRLNRGQAS LTVSVMERAD VGGPRNCAGV ASNYLASCTP GSILRVSLRQ  901ANPDFRLPDE SCSHPIIMVA AGSGIAPFRA FVQERSVRQK EGIILPPAFL FFGCRRADLD  961DLYREELDAF EEQGVVTLFR AFSRAQSESH GCKYVQDLLW MERVRVKTLW GQDAKVFVCG 1021SVRMNEGVKA IISKIVSPTP TEELARRYIA ETFI CYPX A. oryzae ATCC42149Uniprot Accession No. Q2UNA2 >gi|121938553|sp|Q2UNA2|Q2UNA2_ASPOR Cytochrome P450SEQ ID NO: 35    1MSTPKAEPVP IPGPRGVPLM GNILDIESEI PLRSLEMMAD TYGPIYRLTT FGFSRCMISS   61HELAAEVFDE ERFTKKIMAG LSELRHGIHD GLFTAHMGEE NWEIAHRVLM PAFGPLNIQN  121MFDEMHDIAT QLVMKWARQG PKQKIMVTDD FTRLTLDTIA LCAMGTRFNS FYSEEMHPFV  181DAMVGMLKTA GDRSRRPGLV NNLPTTENNK YWEDIDYLRN LCKELVDTRK KNPTDKKDLL  241NALINGRDPK TGKGMSYDSI IDNMITFLIA GHETTSGSLS FAFYNMLKNP QAYQKAQEEV  301DRVIGRRRIT VEDLQKLPYI TAVMRETLRL TPTAPAIAVG PHPTKNHEDP VTLGNGKYVL  361GKDEPCALLL GKIQRDPKVY GPDAEEFKPE RMLDEHFNKL PKHAWKPFGN GMRACIGRPF  421AWQEALLVIA MLLQNFNFQM DDPSYNIQLK QTLTIKPNHF YMRAALREGL DAVHLGSALS  481ASSSEHADHA AGHGKAGAAK KGADLKPMHV YYGSNTGTCE AFARRLADDA TSYGYSAEVE  541SLDSAKDSIP KNGPVVFITA SYEGQPPDNA AHFFEWLSAL KGDKPLDGVN YAVFGCGHHD  601WQTTFYRIPK EVNRLVGENG ANRLCEIGLA DTANADIVTD FDTWGETSFW PAVAAKFGSN  661TQGSQKSSTF RVEVSSGHRA TTLGLQLQEG LVVENTLLTQ AGVPAKRTIR FKLPTDTQYK  721CGDYLAILPV NPSTVVRKVM SRFDLPWDAV LRIEKASPSS SKHISIPMDT QVSAYDLFAT  781YVELSQPASK RDLAVLADAA AVDPETQAEL QAIASDPARF AEISQKRISV LDLLLQYPSI  841NLAIGDFVAM LPPMRVRQYS ISSSPLVDPT ECSITFSVLK APSLAALTKE DEYLGVASTY  901LSELRSGERV QLSVRPSHTG FKPPTELSTP MIMACAGSGL APFRGFVMDR AEKIRGRRSS  961GSMPEQPAKA ILYAGCRTQG KDDIHADELA EWEKIGAVEV RRAYSRPSDG SKGTHVQDLM 1021MEDKKELIDL FESGARIYVC GTPGVGNAVR DSIKSMFLER REEIRRIAKE KGEPVSDDDE 1081ETAFEKFLDD MKTKERYTTD IFA CYP505A1 F. oxysporumUniprot Accession No. Q9Y8G7 >gi|22653677|sp|Q9Y8G7.1|C505_FUSOX RecName: Full =Bifunctional  P-450:NADPH-P450 reductase; AltName: Full =Cytochrome P450foxy;  AltName: Full =Fatty acid omega-hydroxylase; Includes: RecName:  Full =Cytochrome P450 505; Includes: RecName: Full =NADPH--cytochrome P450 reductase SEQ ID NO: 36    1maesvpipep pgyplignlg eftsnplsdl nrladtygpi frlrlgakap ifvssnslin   61evcdekrfkk tlksvlsqvr egvhdglfta fedepnwgka hrilvpafgp lsirgmfpem  121hdiatqlcmk farhgprtpi dtsdnftrla ldtlalcamd frfysyykee lhpfieamgd  181fltesgnrnr rppfapnfly raanekfygd ialmksvade vvaarkasps drkdllaaml  241ngvdpqtgek lsdenitnql itfliaghet tsgtlsfamy qllknpeays kvqkevdevv  301grgpvlvehl tklpyisavl retlrinspi tafgleaidd tflggkylvk kgeivtalls  361rghvdpvvyg ndadkfiper mlddefarin keypncwkpf gngkracigr pfawqeslla  421mvvlfqnfnf tmtdpnyale ikqtltikpd hfyinatlrh gmtptelehv lagngatsss  481thnikaaanl dakagsgkpm aifygsnsgt cealanrlas dapshgfsat tvgpldqakq  541nlpedrpvvi vtasyeggpp snaahfikwm edldgndmek vsyavfacgh hdwvetfhri  601pklvdstlek rggtrlvpmg sadaatsdmf sdfeawediv lwpglkekyk isdeesggqk  661gllvevstpr ktslrqdvee alvvaektlt ksgpakkhie iqlpsamtyk agdylailpl  721npkstvarvf rrfslawdsf lkiqsegptt lptnvaisaf dvfsayvels qpatkrnila  781laeatedkdt iqelerlagd ayqaeispkr vsvldllekf pavalpissy lamlppmrvr  841gysissspfa dpskltltys lldapslsgq grhvgvatnf lshltagdkl hvsvrassea  901fhlpsdaekt piicvaagtg laplrgfiqe raamlaagrt lapallffgc rnpeiddlya  961eeferwekmg avdvrraysr atdksegcky vqdrvyhdra dvfkvwdqga kvficgsrei 1021gkavedvcvr laiekaqqng rdvteemara wfersrnerf atdvfd CYPX G. moniliformisGenBank Accession No. AAG27132>gi|11035011|gb|AAG27132.11 Fum6p [Fusarium verticillioides]SEQ ID NO: 37    1MSATALFTRR SVSTSNPELR PIPGPKPLPL LGNLFDFDFD NLTKSLGELG KIHGPIYSIT   61FGASTEIMVT SREIAQELCD ETRFCKLPGG ALDVMKAVVG DGLFTAETSN PKWAIAHRII  121TPLFGAMRIR GMFDDMKDIC EQMCLRWARF GPDEPLNVCD NMTKLTLDTI ALCTIDYRFN  181SFYRENGAAH PFAEAVVDVM TESFDQSNLP DFVNNYVRFR AMAKFKRQAA ELRRQTEELI  241AARRQNPVDR DDLLNAMLSA KDPKTGEGLS PESIVDNLLT FLIAGHETTS SLLSFCFYYL  301LENPHVLRRV QQEVDTVVGS DTITVDHLSS MPYLEAVLRE TLRLRDPGPG FYVKPLKDEV  361VAGKYAVNKD QPLFIVFDSV HRDQSTYGAD ADEFRPERML KDGFDKLPPC AWKPFGNGVR  421ACVGRPFAMQ QAILAVAMVL HKFDLVKDES YTLKYHVTMT VRPVGFTMKV RLRQGQRATD  481LAMGLHRGHS QEASAAASPS RASLKRLSSD VNGDDTDHKS QIAVLYASNS GSCEALAYRL  541AAEATERGFG IRAVDVVNNA IDRIPVGSPV ILITASYNGE PADDAQEFVP WLKSLESGRL  601NGVKFAVFGN GHRDWANTLF AVPRLIDSEL ARCGAERVSL MGVSDTCDSS DPFSDFERWI  661DEKLFPELET PHGPGGVKNG DRAVPRQELQ VSLGQPPRIT MRKGYVRAIV TEARSLSSPG  721VPEKRHLELL LPKDFNYKAG DHVYILPRNS PRDVVRALSY FGLGEDTLIT IRNTARKLSL  781GLPLDTPITA TDLLGAYVEL GRTASLKNLW TLVDAAGHGS RAALLSLTEP ERFRAEVQDR  841HVSILDLLER FPDIDLSLSC FLPMLAQIRP RAYSFSSAPD WKPGHATLTY TVVDFATPAT  901QGINGSSKSK AVGDGTAVVQ RQGLASSYLS SLGPGTSLYV SLHRASPYFC LQKSTSLPVI  961MVGAGTGLAP FRAFLQERRM AAEGAKQRFG PALLFFGCRG PRLDSLYSVE LEAYETIGLV 1021QVRRAYSRDP SAQDAQGCKY VTDRLGKCRD EVARLWMDGA QVLVCGGKKM ANDVLEVLGP 1081MLLEIDQKRG ETTAKTVVEW RARLDKSRYV EEVYV CYP505A7 G. zeae PH1GenBank Accession No. EAA67736 >gi|42544893|gb|EAA67736.1|C505_FUSOX Bifunctional P-450:NADPH-P450reductase (Fatty acid omega-hydroxylase) (P450foxy) [Gibberella zeae PH-1] SEQ ID NO: 38    1MAESVPIPEP PGYPLIGNLG EFKTNPLNDL NRLADTYGPI FRLHLGSKTP TFVSSNAFIN   61EVCDEKRFKK TLKSVLSVVR EGVHDGLFTA FEDEPNWGKA HRILIPAFGP LSIRNMFPEM  121HEIANQLCMK LARHGPHTPV DASDNFTRLA LDTLALCAMD FRFNSYYKEE LHPFIEAMGD  181FLLESGNRNR RPAFAPNFLY RAANDKFYAD IALMKSVADE VVATRKQNPT DRKDLLAAML  241EGVDPQTGEK LSDDNITNQL ITFLIAGHET TSGTLSFAMY HLLKNPEAYN KLQKEIDEVI  301GRDPVTVEHL TKLPYLSAVL RETLRISSPI TGFGVEAIED TFLGGKYLIK KGETVLSVLS  361RGHVDPVVYG PDAEKFVPER MLDDEFARLN KEFPNCWKPF GNGKRACIGR PFAWQESLLA  421MALLFQNFNF TQTDPNYELQ IKQNLTIKPD NFFFNCTLRH GMTPTDLEGQ LAGKGATTSI  481ASHIKAPAAS KGAKASNGKP MAIYYGSNSG TCEALANRLA SDAAGHGFSA SVIGTLDQAK  541QNLPEDRPVV IVTASYEGQP PSNAAHFIKW MEDLAGNEME KVSYAVFGCG HHDWVDTFLR  601IPKLVDTTLE QRGGTRLVPM GSADAATSDM FSDFEAWEDT VLWPSLKEKY NVTDDEASGQ  661RGLLVEVTTP RKTTLRQDVE EALVVSEKTL TKTGPAKKHI EIQLPSGMTY KAGDYLAILP  721LNPRKTVSRV FRRFSLAWDS FLKIQSDGPT TLPINIAISA FDVFSAYVEL SQPATKRNIL  781ALSEATEDKA TIQELEKLAG DAYQEDVSAK KVSVLDLLEK YPAVALPISS YLAMLPPMRV  841RQYSISSSPF ADPSKLTLTY SLLDAPSLSG QGRHVGVATN FLSQLIAGDK LHISVRASSA  901AFHLPSDPET TPIICVAAGT GLAPFRGFIQ ERAAMLAAGR KLAPALLFFG CRDPENDDLY  961AEELARWEQM GAVDVRRAYS RATDKSEGCK YVQDRIYHDR ADVFKVWDQG AKVFICGSRE 1021IGKAVEDICV RLAMERSEAT QEGKGATEEK AREWFERSRN ERFATDVFD CYP505C2G. zeae PH1a GenBank Accession No. EAA77183 >gi|42554340|gb|EAA77183.1|hypothetical protein FG07596.1  [Gibberella zeae PH-1] SEQ ID NO: 39   1 MAIKDGGKKS GQIPGPKGLP VLGNLFDLDL SDSLTSLINI GQKYAPIFSL ELGGHREVMI  61 CSRDLLDELC DETRFHKIVT GGVDKLRPLA GDGLFTAQHG NHDWGIAHRI LMPLFGPLKI 121 REMFDDMQDV SEQLCLKWAR LGPSATIDVA NDFTRLTLDT IALCTMGYRF NSFYSNDKMH 181 PFVDSMVAAL IDADKQSMFP DFIGACRVKA LSAFRKHAAI MKGTCNELIQ ERRKNPIEGT 241 DLLTAMMEGK DPKTGEGMSD DLIVQNLITF LIAGHETTSG LLSFAFYYLL ENPHTLEKAR 301 AEVDEVVGDQ ALNVDHLTKM PYVNMILRET LRLMPTAPGF FVTPHKDEII GGKYAVPANE 361 SLFCFLHLIH RDPKVWGADA EEFRPERMAD EFFEALPKNA WKPFGNGMRG CIGREFAWQE 421 AKLITVMILQ NFELSKADPS YKLKIKQSLT IKPDGFNMHA KLRNDRKVSG LFKAPSLSSQ 481 QPSLSSRQSI NAINAKDLKP ISIFYGSNTG TCEALAQKLS ADCVASGFMP SKPLPLDMAT 541 KNLSKDGPNI LLAASYDGRP SDNAEEFTKW AESLKPGELE GVQFAVFGCG HKDWVSTYFK 601 IPKILDKCLA DAGAERLVEI GLTDASTGRL YSDFDDWENQ KLFTELSKRQ GVTPTDDSHL 661 ELNVTVIQPQ NNDMGGNFKR AEVVENTLLT YPGVSRKHSL LLKLPKDMEY TPGDHVLVLP 721 KNPPQLVEQA MSCFGVDSDT ALTISSKRPT FLPTDTPILI SSLLSSLVEL SQTVSRTSLK 781 RLADFADDDD TKACVERIAG DDYTVEVEEQ RMSLLDILRK YPGINMPLST FLSMLPQMRP 841 RTYSFASAPE WKQGHGMLLF SVVEAEEGTV SRPGGLATNY MAQLRQGDSI LVEPRPCRPE 901 LRTTMMLPEP KVPIIMIAVG AGLAPFLGYL QKRFLQAQSQ RTALPPCTLL FGCRGAKMDD 961 ICRAQLDEYS RAGVVSVHRA YSRDPDSQCK YVQGLVTKHS ETLAKQWAQG AIVMVCSGKK1021 VSDGVMNVLS PILFAEEKRS GMTGADSVDV WRQNVPKERM ILEVFG CYP505A5M. grisea 70-15 synGenBank Accession No. XP 365223 >gi|145601517|ref|XP_365223.2|hypothetical protein MGG 01925  [Magnaporthe oryzae 70-15] SEQ ID NO: 40   1 MFFLSSSLAY MAATQSRDWA SFGVSLPSTA LGRHLQAAMP FLSEENHKSQ GTVLIPDAQG  61 PIPFLGSVPL VDPELPSQSL QRLARQYGEI YRFVIPGRQS PILVSTHALV NELCDEKRFK 121 KKVAAALLGL REAIHDGLFT AHNDEPNWGI AHRILMPAFG PMAIKGMFDE MHDVASQMIL 181 KWARHGSTTP IMVSDDFTRL TLDTIALCSM GYRFNSFYHD SMHEFIEAMT CWMKESGNKT 241 RRLLPDVFYR TTDKKWHDDA EILRRTADEV LKARKENPSG RKDLLTAMIE GVDPKTGGKL 301 SDSSIIDNLI TFLIAGHETT SGMLSFAFYL LLKNPTAYRK AQQEIDDLCG REPITVEHLS 361 KMPYITAVLR ETLRLYSTIP AFVVEAIEDT VVGGKYAIPK NHPIFLMIAE SHRDPKVYGD 421 DAQEFEPERM LDGQFERRNR EFPNSWKPFG NGMRGCIGRA FAWQEALLIT AMLLQNFNFV 481 MHDPAYQLSI KENLTLKPDN FYMRAILRHG MSPTELERSI SGVAPTGNKT PPRNATRTSS 541 PDPEDGGIPM SIYYGSNSGT CESLAHKLAV DASAQGFKAE TVDVLDAANQ KLPAGNRGPV 601 VLITASYEGL PPDNAKHFVE WLENLKGGDE LVDTSYAVFG CGHQDWTKTF HRIPKLVDEK 661 LAEHGAVRLA PLGLSNAAHG DMFVDFETWE FETLWPALAD RYKTGAGRQD AAATDLTAAL 721 SQLSVEVSHP RAADLRQDVG EAVVVAARDL TAPGAPPKRH MEIRLPKTGG RVHYSAGDYL 781 AVLPVNPKST VERAMRRFGL AWDAHVTIRS GGRTTLPTGA PVSAREVLSS YVELTQPATK 841 RGIAVLAGAV TGGPAAEQEQ AKAALLDLAG DSYALEVSAK RVGVLDLLER FPACAVPFGT 901 FLALLPPMRV RQYSISSSPL WNDEHATLTY SVLSAPSLAD PARTHVGVAS SYLAGLGEGD 961 HLHVALRPSH VAFRLPSPET PVVCVCAGSG MAPFRAFAQE RAALVGAGRK VAPLLLFFGC1021 REPGVDDLYR EELEGWEAKG VLSVRRAYSR RTEQSEGCRY VQDRLLKNRA EVKSLWSQDA1081 KVFVCGSREV AEGVKEAMFK VVAGKEGSSE EVQAWYEEVR NVRYASDIFD CYP505A2N. crassa OR74 AGenBank Accession No. XP 961848 >gi|85104987|ref|XP_961848.1|bifunctional P-450:NADPH-P450 reductase [Neurospora crassa OR74A]SEQ ID NO: 41    1MSSDETPQTI PIPGPPGLPL VGNSFDIDTE FPLGSMLNFA DQYGEIFRLN FPGRNTVFVT   61SQALVHELCD EKRFQKTVNS ALHEIRHGIH DGLFTARNDE PNWGIAHRIL MPAFGPMAIQ  121NMFPEMHEIA SQLALKWARH GPNQSIKVTD DFTRLTLDTI ALCSMDYRFN SYYHDDMHPF  181IDAMASFLVE SGNRSRRPAL PAFMYSKVDR KFYDDIRVLR ETAEGVLKSR KEHPSERKDL  241LTAMLDGVDP KTGGKLSDDS IIDNLITFLI AGHETTSGLL SFAFVQLLKN PETYRKAQKE  301VDDVCGKGPI KLEHMNKLHY IAAVLRETLR LCPTIPVIGV ESKEDTVIGG KYEVSKGQPF  361ALLFAKSHVD PAVYGDTAND FDPERMLDEN FERLNKEFPD CWKPFGNGMR ACIGRPFAWQ  421EALLVMAVCL QNFNFMPEDP NYTLQYKQTL TTKPKGFYMR AMLRDGMSAL DLERRLKGEL  481VAPKPTAQGP VSGQPKKSGE GKPISIYYGS NTGTCETFAQ RLASDAEAHG FTATIIDSLD  541AANQNLPKDR PVVFITASYE GQPPDNAALF VGWLESLTGN ELEGVQYAVF GCGHHDWAQT  601FHRIPKLVDN TVSERGGDRI CSLGLADAGK GEMFTEFEQW EDEVFWPAME EKYEVSRKED  661DNEALLQSGL TVNFSKPRSS TLRQDVQEAV VVDAKTITAP GAPPKRHIEV QLSSDSGAYR  721SGDYLAVLPI NPKETVNRVM RRFQLAWDTN ITIEASRQTT ILPTGVPMPV HDVLGAYVEL  781SQPATKKNIL ALAEAADNAE TKATLRQLAG PEYTEKITSR RVSILDLLEQ FPSIPLPFSS  841FLSLLPPMRV RQYSISSSPL WNPSHVTLTY SLLESPSLSN PDKKHVGVAT SYLASLEAGD  901KLNVSIRPSH KAFHLPVDAD KTPLIMIAAG SGLAPFRGFV QERAAQIAAG RSLAPAMLFY  961GCRHPEQDDL YRDEFDKWES IGAVSVRRAF SRCPESQETK GCKYVGDRLW EDREEVTGLW 1021DRGAKVYVCG SREVGESVKK VVVRIALERQ KMIVEAREKG ELDSLPEGIV EGLKLKGLTV 1081EDVEVSEERA LKWFEGIRNE RYATDVFD CYP97C Oryza sativaGenBank Accession No. ABB47954 >gi|78708979|gb|ABB47954.1|Cytochrome P450 family protein, expressed  [Oryza sativa Japonica Group]SEQ ID NO: 42   1MAAAAAAAVP CVPFLCPPPP PLVSPRLRRG HVRLRLRPPR SSGGGGGGGA GGDEPPITTS  61WVSPDWLTAL SRSVATRLGG GDDSGIPVAS AKLDDVRDLL GGALFLPLFK WFREEGPVYR 121LAAGPRDLVV VSDPAVARHV LRGYGSRYEK GLVAEVSEFL FGSGFAIAEG ALWTVRRRSV 181VPSLHKRFLS VMVDRVFCKC AERLVEKLET SALSGKPVNM EARFSQMTLD VIGLSLFNYN 241FDSLTSDSPV IDAVYTALKE AELRSTDLLP YWKIDLLCKI VPRQIKAEKA VNIIRNTVED 301LITKCKKIVD AENEQIEGEE YVNEADPSIL RFLLASREEV TSVQLRDDLL SMLVAGHETT 361GSVLTWTIYL LSKDPAALRR AQAEVDRVLQ GRLPRYEDLK ELKYLMRCIN ESMRLYPHPP 421VLIRRAIVDD VLPGNYKIKA GQDIMISVYN IHRSPEVWDR ADDFIPERFD LEGPVPNETN 481TEYRFIPFSG GPRKCVGDQF ALLEAIVALA VVLQKMDIEL VPDQKINMTT GATIHTTNGL 541YMNVSLRKVD REPDFALSGS R Chimeric heme enzyme C2G9 SEQ ID NO: 43MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLIQKWARLNPNEAVDVPGDMTRLTLDTIGLCGENYRENSYYRETPHPFINSMVRALDEAMHQMQRLDVQDKLMVRTKRQFRYDIQTMESLVDRMIAERKANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALHEATLVLGMILKYFTLIDHENYELDIKQTLTLKPGDFHISVQSRHQEAIHADVQAAE Chimeric heme enzyme X7 SEQ ID NO: 44MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERRANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQA Chimeric heme enzyme X7-12 SEQ ID NO: 45MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDEERFDKSIEGALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERRANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQA Chimeric heme enzyme C2E6 SEQ ID NO: 46MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERKANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPST Chimeric heme enzyme X7-9 SEQ ID NO: 47MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFREDFPGVSSVFVSGHNLVAEVCDEERFDKSIEGALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERRANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQA Chimeric heme enzyme C2B12 SEQ ID NO: 48MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFREDFPGVSSVFVSGHNLVAEVCDEERFDKSIEGALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERKANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFATYFLLKHPDKLKKAYEEVDRVLTDAAPTYKQVLELTYIRMILNESLRLWPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQA Chimeric heme enzyme TSP234 SEQ ID NO: 49MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERKANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQA

1. A reaction mixture for producing an aziridination product, thereaction mixture comprising of an olefinic substrate, a nitreneprecursor, and a heme enzyme.
 2. The reaction mixture of claim 1,wherein the olefinic substrate is represented by a structure of FormulaI:

wherein: R^(1a), R^(1b), and R² are independently selected from thegroup consisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl, aryl, heteroaryl,C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl, —Y¹-heteroaryl,—Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl; Y¹ is C₁₋₈alkylene; eachR^(1a), R^(1b), and R² is optionally substituted with from 1 to 5substituents independently selected from the group consisting ofC₁₋₃alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo,cyano, and halogen; wherein each aryl contains between 6-14 carbonatoms, each heteroaryl group has from 5 to 8 ring atoms and from 1-3heteroatoms selected from N, O and S, and each heterocyclyl group hasfrom 1-3 heteroatoms selected from N, O and S.
 3. The reaction mixtureof claim 2, wherein R^(1a), R^(1b), and R² are independently selectedfrom the group consisting of H, C₁₋₁₈alkyl, aryl, heteroaryl,C₁₋₁₂cycloalkyl, and C₃₋₁₀heterocyclyl, and each R^(1a), R^(1b), and R²is optionally substituted with from 1 to 5 substituents independentlyselected from the group consisting of C₁₋₃alkyl, alkoxy, and halogen. 4.The reaction mixture of claim 1, wherein the nitrene precursor has aformula selected from the group consisting of:

wherein R³ is selected from the group consisting of C₁₋₁₈ alkyl,C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl, C₃₋₁₀heterocyclyl,—SO₂R^(a), —COR^(a), —CO₂R^(b), —PO₃R^(b)R^(c), and —CONR^(b)R^(c); X¹is independently selected from the group consisting of H and sodium, andX² is independently selected from the group consisting of halogen,—SO₂R^(a), —CO₂R^(b), —PO₃R^(b)R^(c), optionally X¹ and X² can be takentogether to form iodinane; R^(a) is independently selected from thegroup consisting of C₁₋₈alkyl, hydroxy, C₁₋₈alkoxy, C₃₋₁₂cycloalkyl,aryl, heteroaryl, and C₃₋₈heterocyclyl; R^(b) and R^(c) areindependently selected from the group consisting of C₁₋₈alkyl,C₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl; wherein withineach R³, R^(a), R^(b), and R^(c) can be optionally substituted with from1-5 R^(d) substituents; each R^(d) is independently selected from thegroup consisting of C₁₋₃alkyl, halogen, and hydroxy; and wherein eacharyl contains between 6-14 carbon atoms, each heteroaryl group has from5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S,and each heterocyclyl group has from 1-3 heteroatoms selected from N, Oand S.
 5. The reaction mixture of claim 4, wherein the nitrene precursoris selected from the group consisting of:


6. The reaction mixture of claim 5, wherein the nitrene precursor is


7. The reaction mixture of claim 1, wherein the aziridination product isproduced in vitro.
 8. The reaction mixture of claim 7, wherein thereaction mixture further comprises a reducing agent.
 9. The reactionmixture of claim 8, wherein the reducing agent is NADPH.
 10. Thereaction mixture of claim 1, wherein the heme enzyme is localized withina whole cell and the aziridination product is produced in vivo.
 11. Thereaction mixture of claim 10, wherein the whole cell is a bacterial cellor a yeast cell.
 12. The reaction mixture of claim 1, wherein theaziridination product is produced under anaerobic conditions.
 13. Thereaction mixture of claim 1, wherein the heme enzyme is a variantthereof comprising a mutation at the axial position of the hemecoordination site.
 14. The reaction mixture of claim 13, wherein theheme enzyme comprises a serine mutation at the axial position of theheme coordination site.
 15. The reaction mixture of claim 1, wherein theheme enzyme is a cytochrome P450 enzyme or a variant thereof.
 16. Thereaction mixture of claim 15, wherein the cytochrome P450 enzyme is aP450 BM3 enzyme or a variant thereof.
 17. The reaction mixture of claim16, wherein the P450 BM3 enzyme comprises an axial ligand mutation C400Sand one or more mutations selected from the group consisting of V78,F87, P142, T175, A184, S226, H236, E252, I263, T268, A290, A328, L353,I366, L437, T438, and E442 relative to the amino acid sequence set forthin SEQ ID NO:1 (SEQ ID NO: 50).
 18. The reaction mixture of claim 17,wherein the P450 BM3 enzyme comprises an axial ligand mutation C400S andmutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F,T268A, A290V, A328V, L353V, I366V, L437V, T438S, and E442K relative tothe amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 51). 19.The reaction mixture of claim 16, wherein the P450 BM3 enzyme comprisesan axial ligand mutation C400S and one or more mutations selected fromthe group consisting of L75, V78, F87, P142, T175, L181, A184, S226,H236, E252, I263, T268, A290, L353, I366, and E442 relative to the aminoacid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 52).
 20. Thereaction mixture of claim 19, wherein the P450 BM3 enzyme comprises anaxial ligand mutation C400S and mutations L75A, V87A, F87V, P142S,T175I, L181A, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V,I366V, and E442K relative to the amino acid sequence set forth in SEQ IDNO:1 (SEQ ID NO: 53).
 21. The reaction mixture of claim 1, wherein theaziridination product is an aziridine compound according to Formula III:

wherein R^(1a), R^(1b), and R² are independently selected from the groupconsisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl, aryl, heteroaryl,C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl, —Y¹-heteroaryl,—Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl; Y¹ is C₁₋₈alkylene; eachR^(1a), R^(1b), and R² is optionally substituted with from 1 to 5substituents independently selected from the group consisting ofC₁₋₃alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo,cyano, and halogen; R³ is selected from the group consisting of C₁₋₁₈alkyl, C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b), —PO₃R^(b)R^(c), and—CONR^(b)R^(c); R^(a) is independently selected from the groupconsisting of C₁₋₈alkyl, hydroxy, C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl,heteroaryl, and C₃₋₈heterocyclyl; R^(b) and R^(c) are independentlyselected from the group consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl,heteroaryl, and C₃₋₈heterocyclyl; wherein within each R³, R^(a), R^(b),and R^(c) can be optionally substituted with from 1-5 R^(d)substituents; each R^(d) is independently selected from the groupconsisting of C₁₋₃alkyl, halogen, and hydroxy; and wherein each arylcontains between 6-14 carbon atoms, each heteroaryl group has from 5 to10 ring atoms and from 1-3 heteroatoms selected from N, O and S, andeach heterocyclyl group has from 1-3 heteroatoms selected from N, O andS.
 22. The reaction mixture of claim 21, wherein R^(1a) and R^(1b) areindependently selected from the group consisting of H, C₁₋₈alkyl, aryl,heteroaryl, C₁₋₁₂cycloalkyl, and C₃₋₁₀heterocyclyl; R² is selected fromthe group consisting of H and C₁₋₈ alkyl; each R^(1a), R^(1b), and R² isoptionally substituted with from 1 to 3 substituents independentlyselected from the group consisting of C₁₋₃alkyl, alkoxy, and halogen;and R³ is selected from the group consisting of —SO₂R^(a), —COR^(a),—CO₂R^(b), —PO₃R^(b)R^(c), and —CONR^(b)R^(c), R^(a) is independentlyselected from the group consisting of C₃₋₁₂cycloalkyl, aryl, heteroaryl,and C₃₋₈heterocyclyl; R^(b) and R^(c) are independently selected fromthe group consisting of C₃₋₁₂cycloalkyl, aryl, heteroaryl, andC₃₋₈heterocyclyl; wherein within each R³, R^(a), R^(b), and R^(c) can beoptionally substituted with from 1-2 R^(d) substituents; and each R^(d)is independently selected from the group consisting of C₁₋₃alkyl,halogen, and hydroxy.
 23. The reaction mixture of claim 1, wherein theaziridination product is an amido-alcohol compound according to FormulaIIIa:

wherein R^(1a), R^(1b), and R² are independently selected from the groupconsisting of H, C₁₋₁₈alkyl, C₁₋₈heteroalkyl, aryl, heteroaryl,C₁₋₁₂cycloalkyl, C₃₋₁₀heterocyclyl, —Y¹-aryl, —Y¹-heteroaryl,—Y¹—C₁₋₁₂cycloalkyl and —Y¹—C₃₋₁₀heterocyclyl; Y¹ is C₁₋₈alkylene; eachR^(1a), R^(1b), and R² is optionally substituted with from 1 to 5substituents independently selected from the group consisting ofC₁₋₃alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo,cyano, and halogen; R³ is selected from the group consisting of C₁₋₁₈alkyl, C₁₋₈heteroalkyl, C₃₋₁₂cycloalkyl, aryl, heteroaryl,C₃₋₁₀heterocyclyl, —SO₂R^(a), —COR^(a), —CO₂R^(b), —PO₃R^(b)R^(c), and—CONR^(b)R^(c); R^(a) is independently selected from the groupconsisting of C₁₋₈alkyl, hydroxy, C₁₋₈alkoxy, C₃₋₁₂cycloalkyl, aryl,heteroaryl, and C₃₋₈heterocyclyl; R^(b) and R^(c) are independentlyselected from the group consisting of C₁₋₈alkyl, C₃₋₁₂cycloalkyl, aryl,heteroaryl, and C₃₋₈heterocyclyl; wherein within each R³, R^(a), R^(b),and R^(c) can be optionally substituted with from 1-5 R^(d)substituents; each R^(d) is independently selected from the groupconsisting of C₁₋₃alkyl, halogen, and hydroxy; and wherein each arylcontains between 6-14 carbon atoms, each heteroaryl group has from 5 to10 ring atoms and from 1-3 heteroatoms selected from N, O and S, andeach heterocyclyl group has from 1-3 heteroatoms selected from N, O andS.
 24. The reaction mixture of claim 23, wherein R^(1a) and R^(1b), areindependently selected from the group consisting of H, C₁₋₈alkyl, aryl,heteroaryl; R² is selected from the group consisting of H, and C₁₋₈alkyl, each R^(1a), R^(1b), and R² is optionally substituted with from 1to 3 substituents independently selected from the group consisting ofC₁₋₃alkyl, alkoxy and halogen; and R³ is selected from the groupconsisting of —SO₂R^(a), —COR^(a), —CO₂R^(b), —PO₃R^(b)R^(c), and—CONR^(b)R^(c), R^(a) is independently selected from the groupconsisting of C₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl;R^(b) and R^(c) are independently selected from the group consisting ofC₃₋₁₂cycloalkyl, aryl, heteroaryl, and C₃₋₈heterocyclyl; wherein withineach R³, R^(a), R^(b), and R^(c) can be optionally substituted with from1-2 R^(d) substituents; and each R^(d) is independently selected fromthe group consisting of C₁₋₃alkyl, halogen, and hydroxy.
 25. Thereaction mixture of claim 1, wherein the reaction produces a pluralityof aziridination products.
 26. The reaction mixture of claim 25, whereinthe plurality of aziridination products has a % ee_(S) of from about−99% to about 99%.
 27. The reaction mixture of claim 25, wherein theplurality of aziridination products has a % ee_(S) of from about −86% toabout 86%.
 28. The reaction mixture of claim 25, wherein the pluralityof aziridination products has a Z:E ratio of from about 1:99 to about99:1.
 29. The reaction mixture of claim 25, wherein the reaction is atleast 30% to at least 90% diastereoselective. 30-49. (canceled)